autographa californica

Abstract

The present invention provides noctuid dsx splice cassettes for expressing a gene of interest on a gender-specific basis, gene expression systems for conferring self-limiting traits to transformed noctuids, and methods of inhibiting noctuid populations and reducing, inhibiting or eliminating crop damage caused by noctuid insects.

Description

[0001] This application is a divisional application of the international application number PCT / GB2019 / 050897, international application date March 28, 2019, Chinese application number 201980034747.X, entitled "Self-limiting Noctuid Moth".

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Provisional Patent Application 62 / 649,912, filed March 29, 2018, the entire contents of which are incorporated herein by reference.

[0004] sequence list reference

[0005] The “Sequence List” (labeled below) is incorporated herein by reference in its entirety as a text file. A copy of the text file of the Sequence List, which is filed with this application, is labeled “Sequence List”, is 131,925 bytes in size, and was created on March 27, 2019; the entire Sequence List is incorporated herein by reference. Background Technology

[0006] Noctuid moths, or the family Noctuidae, are known by many common names, including cutworm, armyworm, and flying moth. The family Noctuidae encompasses over 1,000 genera and more than 11,000 species. Several genera and species within Noctuidae cause massive crop damage each year, resulting in billions of dollars in losses. Several genera, including *Spodoptera*, *Helicoverpa*, *Chrysodeixis*, *Anticarsia*, *Peridroma*, and *Heliothis*, are among the leading insects causing global crop losses. Important species include, for example, the fall armyworm (Spodoptera frugiperda), the beet armyworm (Spodoptera exigua), the African cotton leafworm (Spodoptera littoralis), the cotton bollworm (Helicoverpa armigera), the leaf-eating moth (Peridromasaucia), the grain moth (Helicoverpa zea); other common names include cotton bollworm and tomato leaf-eating moth, the soybean moth (Chrysodeixis includens), the velvet bean caterpillar (Anticarsiagemmatalis), and the tobacco moth (Heliothisvirescens).

[0007] The fall armyworm affects a variety of crops, including corn, rice, cotton, sugarcane, and sorghum. Each female moth lays approximately 2,000 eggs per swarm, with each moth producing about 1,000 eggs. The larvae hatched from these eggs then consume the crops. Several generations of armyworms may reproduce each year.

[0008] Attempts to control noctuid moths have primarily involved the use of pesticides. However, insects have developed resistance to pesticides such as pyrethroids, carbamates, and organophosphates. Other attempts to control insects include the use of genetically modified crops, such as plants expressing insecticidal proteins (e.g., Cry1Fa) from microorganisms like Bacillus thuringiensis (Bt crops). However, insects have also developed resistance to Bt crops.

[0009] There is a great need in this field to develop a solution that suppresses noctuid moth populations in a manner different from existing methods of action, thereby reducing reliance on current practices and thus mitigating resistance, and potentially reversing the trend of pesticide resistance in insects.

[0010] Insect sterilization (SIT), where insects are irradiated and released to mate with wild insects of the same species, is effective in suppressing insect populations (Insect Sterility, Dyck, VAJ Hendrichs, J. Robinson, Eds; Springer Netherlands, 2005). A biological alternative to this SIT method is the use of a self-limiting gene, in which insects are genetically engineered to contain a repressible gene that, when expressed, causes the insect to die. In the self-limiting gene method, male insects carrying the self-limiting gene are released into wild insects of the same species; the offspring inherit the self-limiting gene and cannot survive to adulthood.

[0011] Recent developments in the self-limiting approach utilize the sex-specific expression of genes and allow for the modification of insect species in which only females express the self-limiting gene (WO 2007 / 091099). When male self-limiting insects are released into wild populations, all offspring inherit the self-limiting gene, but due to sex-specific expression, only the females fail to survive to adulthood. This development enables the mass breeding and release of self-limiting male insects. In many cases, large-scale rearing and physical separation of the sexes are impractical, or at least labor-intensive.

[0012] The sex-specific splicing gene (dsx) has been used in Dipteran species to create sex-specific splicing (WO 2018 / 029534) and in Lepidopteran species (Jin, L. et al. (2013) ACSSynth. Biol. 2(3): 160-166; Tan, A et al. (2013) Proc. Natl. Acad. Sci. USA 110(17): 6766-6770). Although there is some conservation between Dipteran and Lepidopteran dsx, the sex-specific splicing mechanism in Lepidopterans appears to differ from that in other insects. Diptera, Coleoptera, and Hymenoptera all regulate dsx mRNA precursor splicing via the TRA / TRA2 complex, while Lepidoptera appear to lack TRA homologs and use different genes to determine sex for dsx (Nagaraju, J. et al. (2014) Sex.Devel. 8(1-3): 104-12). Male and female DSX protein isoforms produced by Lepidoptera have the same N-terminal region but differ in the C-terminal portion of the protein, which is necessary for male and female DSX protein isoforms to have different sex-specific functions (Suzuki, MG et al. (2005) Evol.Dev. 7(1): 58-68; Shukla, JN and J. Nagaraju (2010) Insect Niochem.Mol.Biol. 40(9): 672-682; Xu, J. et al. (2017) Insect Biochem.Mol.Biol. 80: 42-51).

[0013] There is a need in this field to develop self-limiting noctuid moths to suppress populations of these insects that severely damage crops and reduce the world’s food supply. Summary of the Invention

[0014] This invention provides a splicing cassette for guiding sex-specific splicing of heterologous polynucleotides encoding functional proteins in arthropods (wherein the coding sequence of the functional protein is defined between a start codon and a stop codon). The cassette comprises at least one exon 2, or a portion thereof, of a noctuid dsx gene; at least one exon 3, or a portion thereof, of a noctuid dsx gene; at least one exon 5, or a portion thereof, of a noctuid dsx gene; at least one intron 2, or a portion thereof, of a noctuid dsx gene; and at least one intron 4, or a portion thereof, of a noctuid dsx gene; wherein (a) a first splicing of the RNA transcript of the heterologous polynucleotide produces a first spliced ​​mRNA product that does not have a continuous open reading frame extending from the start codon to the stop codon; and (b) alternative splicing of the RNA transcript produces an alternatively spliced ​​mRNA product that includes a continuous open reading frame extending from the start codon to the stop codon.

[0015] In some embodiments, the splice cassette comprises at least one exon 2 or a portion thereof of a Noctuidae bisexual (dsx) gene; at least one exon 3 or a portion thereof of a Noctuidae dsx gene; at least one exon 4 or a portion thereof of a Noctuidae dsx gene; at least one exon 5 or a portion thereof of a Noctuidae dsx gene; at least one intron 2 or a portion thereof of a Noctuidae dsx gene; at least one intron 3 or a portion thereof of a Noctuidae dsx gene; at least one intron 4 or a portion thereof of a Noctuidae dsx gene; wherein (a) first splicing of the RNA transcript of the heteropolynucleotide produces a first spliced ​​mRNA product, the first spliced ​​mRNA product not having a continuous open reading frame extending from the start codon to the stop codon; (b) alternative splicing of the RNA transcript produces an alternative spliced ​​mRNA product, which includes a continuous open reading frame extending from the start codon to the stop codon.

[0016] In some embodiments, the box optionally contains at least one exon 3a or a portion thereof of the Noctuidae dsx gene and / or at least one exon 4b or a portion thereof of the Noctuidae dsx gene.

[0017] In some embodiments, the polynucleotide encoding the functional protein is located at the 3' of exons 2 and 3. In other embodiments, the polynucleotide encoding the functional protein is located at the 3' of exons 2, 3, and 5. In still other embodiments, the polynucleotide encoding the functional protein is located at the 3' of exons 2, 3, 3a, 4, 4b, and 5.

[0018] In some embodiments, the primary transcript from the splice cassette in males is spliced, causing translation to terminate at the 5' of the polynucleotide encoding the functional protein, and the functional protein is not translated. In other embodiments, the primary transcript from the splice cassette in males is spliced, causing the polynucleotide encoding the functional protein to be cut off from the primary transcript.

[0019] In some embodiments, exon 2 of the splice cassette comprises a polynucleotide encoding an amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 100% identity with the sequence of SEQ ID NO:71. In some embodiments, exon 2 has a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:71. In other embodiments, exon 2 has a polynucleotide sequence of SEQ ID NO:7, SEQ ID NO:32, or SEQ ID NO:54.

[0020] In some embodiments, exon 3 of the splice cassette comprises a polynucleotide encoding an amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 100% identity with the sequence encoding the amino acid sequence of SEQ ID NO:72. In some embodiments, exon 3 has a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:72. In some embodiments, exon 3 has a core polynucleotide sequence of SEQ ID NO:93, SEQ ID NO:56, or may be divided into two parts (SEQ ID NO:94 and SEQ ID NO:9) and a polynucleotide encoding a lethal, harmful, or sterile (e.g., tTAV or its analogues) protein inserted between these two parts by a linker. Examples of useful linkers include those shown in SEQ ID NO:95 and SEQ ID NO:96 (see [link to relevant documentation]). Figure 19 ).

[0021] In some embodiments, exon 3a of the splice cassette comprises a polynucleotide sequence having 80%, 85%, 90%, 95%, 98%, or 100% identity with the sequence encoding the amino acid sequence of SEQ ID NO:73. In some embodiments, exon 3a has a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:73. In some embodiments, exon 3a has a polynucleotide sequence of SEQ ID NO:12.

[0022] In some embodiments, exon 4 of the splice cassette comprises a polynucleotide having 80%, 85%, 90%, 95%, 98%, or 100% identity with the sequence encoding the amino acid sequence of SEQ ID NO:74. In some embodiments, exon 4 has a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:74. In some embodiments, exon 4 has a polynucleotide sequence of SEQ ID NO:15.

[0023] In some embodiments, exon 4b of the splice cassette contains a polynucleotide sequence having 80%, 85%, 90%, 95%, 98%, or 100% identity with the polynucleotide sequence of SEQ ID NO:14.

[0024] In some embodiments, exon 5 of the splice cassette contains a polynucleotide encoding the amino acid sequence of SEQ ID NO:75. In some embodiments, exon 5 has a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:75. In some embodiments, exon 5 has a polynucleotide sequence of SEQ ID NO:17.

[0025] In some embodiments, the intron 2 of the splice cassette contains a polynucleotide having a sequence having 80%, 85%, 90%, 95%, 98%, or 100% identity with the sequence of SEQ ID NO:55.

[0026] In some embodiments, the intron 3 of the splice cassette contains a polynucleotide having a sequence having 80%, 85%, 90%, 95%, 98%, or 100% identity with the sequence of SEQ ID NO:58.

[0027] In some embodiments, the intron 4 of the splice cassette contains a polynucleotide having a sequence having 80%, 85%, 90%, 95%, 98%, or 100% identity with the sequence of SEQ ID NO:39.

[0028] In some embodiments, the splice cassette includes exon 2 of the Noctuidae dsx family having a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32; exon 3 of the Noctuidae dsx family having a polynucleotide sequence of SEQ ID NO:94, SEQ ID NO:34 or SEQ ID NO:56; exon 5 of the Noctuidae dsx family having a polynucleotide sequence of SEQ ID NO:17; intron 2 having a polynucleotide sequence of SEQ ID NO:55; and intron 4 having a polynucleotide sequence of SEQ ID NO:39 (see also...). Figure 18 ).

[0029] In other embodiments, the splice cassette includes a Noctuidae dsx exon 2 having a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32; a Noctuidae dsx exon 3 having a polynucleotide sequence of SEQ ID NO:94, SEQ ID NO:34 or SEQ ID NO:56; a Noctuidae dsx exon 4 having a polynucleotide sequence of SEQ ID NO:15; a Noctuidae dsx exon 5 having a polynucleotide sequence of SEQ ID NO:17; a Noctuidae dsx intron 2 having a polynucleotide sequence of SEQ ID NO:55; a Noctuidae dsx intron 3 having a polynucleotide sequence of SEQ ID NO:58; and a Noctuidae dsx intron 4 having a polynucleotide sequence of SEQ ID NO:39.

[0030] In some embodiments, the splice cassette includes Noctuidae dsx exon 2 containing a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32; Noctuidae dsx exon 3 containing a polynucleotide sequence of SEQ ID NO:94, SEQ ID NO:34 or SEQ ID NO:56; Noctuidae dsx exon 3a containing a polynucleotide sequence of SEQ ID NO:12; Noctuidae dsx exon 4 containing a polynucleotide sequence of SEQ ID NO:15; Noctuidae dsx exon 4b containing a polynucleotide sequence of SEQ ID NO:14; and Noctuidae dsx exon 5 containing a polynucleotide sequence of SEQ ID NO:17; Noctuidae dsx intron 2 containing a polynucleotide sequence of SEQ ID NO:55; Noctuidae dsx intron 3 containing a polynucleotide sequence of SEQ ID NO:58; and Noctuidae dsx intron 4 containing a polynucleotide sequence of SEQ ID NO:39.

[0031] In some embodiments, the splice cassette includes Noctuidae dsx exon 2 having a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32; Noctuidae dsx exon 3 having a polynucleotide sequence of SEQ ID NO:94, SEQ ID NO:34 or SEQ ID NO:56; Noctuidae dsx exon 3a having a polynucleotide sequence of SEQ ID NO:12; Noctuidae dsx exon 4 having a polynucleotide sequence of SEQ ID NO:15; Noctuidae dsx exon 4b having a polynucleotide sequence of SEQ ID NO:14; and Noctuidae dsx exon 5 having a polynucleotide sequence of SEQ ID NO:17. In other embodiments, the splice cassette includes: Noctuidae dsx exon 2 having a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32; Noctuidae dsx exon 3 having a polynucleotide sequence of SEQ ID NO:94, SEQ ID NO:34 or SEQ ID NO:56; Noctuidae dsx exon 3a having a polynucleotide sequence of SEQ ID NO:12; Noctuidae dsx exon 4 having a polynucleotide sequence of SEQ ID NO:15; Noctuidae dsx exon 4b having a polynucleotide sequence of SEQ ID NO:14; Noctuidae dsx exon 5 having a polynucleotide sequence of SEQ ID NO:17; Noctuidae dsx intron 2 having a polynucleotide sequence of SEQ ID NO:55; Noctuidae dsx intron 3 having a polynucleotide sequence of SEQ ID NO:58; and Noctuidae dsx intron 4 having a polynucleotide sequence of SEQ ID NO:39.

[0032] In other embodiments, the splice cassette includes: Noctuidae dsx exon 2 having a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32; Noctuidae dsx exon 3 having a polynucleotide sequence of SEQ ID NO:94, SEQ ID NO:34 or SEQ ID NO:56; Noctuidae dsx exon 3a having a polynucleotide sequence of SEQ ID NO:12; Noctuidae dsx exon 4 having a polynucleotide sequence of SEQ ID NO:15; Noctuidae dsx; Noctuidae dsx exon 5 having a polynucleotide sequence of SEQ ID NO:17; Noctuidae dsx intron 2 having a polynucleotide sequence of SEQ ID NO:55; Noctuidae dsx intron 3 having a polynucleotide sequence of SEQ ID NO:58; and Noctuidae dsx intron 4 having a polynucleotide sequence of SEQ ID NO:39.

[0033] The box can be used for arthropods such as insects. In some embodiments, the insect is a noctuid moth. Non-limiting examples of this noctuid moth family include insects belonging to the genera *Spodoptera*, *Helicoverpa*, *Chrysodeixis*, *Anticarsia*, *Peridroma*, and *Heliothis*. Specific species include, but are not limited to, the fall armyworm (Spodoptera frugiperda), the beet armyworm (Spodoptera exigua), the African cotton leafworm (Spodoptera littoralis), the cotton bollworm (Helicoverpa armigera), the leaf-spotting moth (Peridroma saucia), the grain moth (Helicoverpa zea), the soybean moth (Chrysodeixis includens), the velvet bean caterpillar (Anticarsia gemmatalis), and the tobacco moth (Heliothis virescens).

[0034] In some embodiments, the cassette has exons and introns derived from the dsx gene of a genus of noctuid moths, including but not limited to *Spodoptera*, *Helicoverpa*, *Chrysodeixis*, *Anticarsia*, *Peridroma*, or *Heliothis*. In some embodiments, the exons and introns are derived from the dsx gene of at least one of *Spodopterafrugiperda*, *Spodoptera exigua*, *Spodoptera littoralis*, *Helicoverpa armigera*, *Peridroma saucia*, *Helicoverpa zea*, *Chrysodeixis includens*, *Anticarsia gemmatalis*, or *Heliothis virescens*.

[0035] In some embodiments, the splice cassette further includes a ubiquitin leader sequence of the 5' polynucleotide encoding the functional protein.

[0036] The present invention also provides a female-specific gene expression system for controlling effector gene expression in arthropods, comprising:

[0037] a. Promoter;

[0038] b. Polynucleotides encoding functional proteins, whose coding sequence is confined between the start codon and the stop codon;

[0039] C. A splicing control polynucleotide that collaborates with the spliceosome in arthropods, capable of mediating the splicing of primary transcripts in an arthropod sex-specific manner, wherein the primary transcript contains exon 2 or a portion thereof of the Noctuidae bisexual (dsx) gene; exon 3 or a portion thereof of the Noctuidae dsx gene; exon 4 or a portion thereof of the Noctuidae dsx gene; exon 5 or a portion thereof of the Noctuidae dsx gene; intron 2 or a portion thereof of the Noctuidae dsx gene; intron 4 or a portion thereof of the Noctuidae dsx gene; optionally, exon 3a or a portion thereof of the Noctuidae dsx gene; optionally, intron 3 or a portion thereof of the Noctuidae dsx gene; and optionally, exon 4b or a portion thereof, thereby forming exon 4b-exon 4 of the Noctuidae dsx gene; wherein:

[0040] (1) The first splicing of a polynucleotide RNA transcript produces a first-spliced ​​mRNA product that does not have a continuous open reading frame extending from the start codon to the stop codon; and

[0041] (2) Alternative splicing of RNA transcripts produces alternatively spliced ​​mRNA products, which include consecutive open reading frames extending from the start codon to the stop codon.

[0042] In some embodiments, the functional protein has lethal, harmful, or sterile effects on arthropods. Examples of functional proteins include, but are not limited to, Hid or its homologs, Reaper (Rpr) or its homologs, Nipp1Dm or its homologs, calmodulin or its homologs, Michelob-X or its homologs, tTAV or its homologs, tTAV2 or its homologs, tTAV3 or its homologs, tTAF or its homologs, medea or its homologs, or nucleases. In other embodiments, the polynucleotide encodes a microRNA toxin, rather than a protein with lethal, harmful, or sterile effects. In some embodiments, the polynucleotide encoding the functional protein encodes tTAV or its homologs, tTAV2 or its homologs, tTAV3 or its homologs, or tTAF or its homologs. Non-limiting examples include proteins having the amino acid sequence of SEQ ID NO:80, SEQ ID NO:97, or SEQ ID NO:98. In some embodiments, the nuclease is FokI or EcoRI.

[0043] The arthropod female-specific gene expression system may further include a 3'UTR or a portion thereof operatively linked to a multinucleotide encoding a functional protein. In some embodiments, the 3'UTR is a P10 3'UTR or a portion thereof.

[0044] In some implementations, the arthropod female-specific gene expression system may further include a ubiquitin leader sequence of a 5' polynucleotide encoding a functional protein.

[0045] In some embodiments of the arthropod female-specific gene expression system, the polynucleotide encoding a functional protein is located at the 3' of exon 2, and within exon 3, such that the polynucleotide encoding the functional protein is flanked by a first portion of exon 3 encoding a polynucleotide 5' of the functional protein, and a second portion of exon 3 encoding a polynucleotide 3' of the functional protein. In a non-limiting example, the first portion has the polynucleotide sequence of SEQ ID NO:94, while the second portion comprises the polynucleotide sequence of SEQ ID NO:9. In other embodiments, the polynucleotide encoding the functional protein is located at the 3' of exons 2, 3, 3a, 4, 4b, and 5.

[0046] In some embodiments, the primary transcript is spliced ​​in males, causing translation to terminate at the 5' end of the polynucleotide encoding the functional protein. In other embodiments, the primary transcript is spliced ​​in males, causing the polynucleotide encoding the functional protein to be cleaved off the primary transcript.

[0047] In some embodiments, exon 2 comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:71. Non-limiting examples of polynucleotides of exon 2 include SEQ ID NO:7 and SEQ ID NO:32.

[0048] In some embodiments, exon 3 comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:72. For example, it may be a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:94, SEQ ID NO:34, or SEQ ID NO:56.

[0049] In some embodiments, exon 3a contains a polynucleotide encoding the amino acid sequence of SEQ ID NO:73. Such an amino acid sequence may be encoded, for example, by the nucleic acid sequence of SEQ ID NO:12.

[0050] In some embodiments, exon 4 comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:74. Such an amino acid sequence may be encoded by, for example, a nucleic acid sequence of SEQ ID NO:15. In some embodiments, exon 4b comprises the polynucleotide sequence of SEQ ID NO:14. In some embodiments, exon 4b and exon 4 are linked to form exon 4b-exon 4, and may have, for example, a polynucleotide sequence of SEQ ID NO:90, SEQ ID NO:91, or SEQ ID NO:92.

[0051] In some embodiments, exon 5 contains a polynucleotide encoding the amino acid sequence of SEQ ID NO:75. Such an amino acid sequence may be encoded by, for example, a nucleic acid sequence of SEQ ID NO:17.

[0052] In some embodiments, intron 2 comprises the polynucleotide sequence of SEQ ID NO:55.

[0053] In some embodiments, intron 3 comprises the polynucleotide sequence of SEQ ID NO:58.

[0054] In some implementations, intron 4 comprises the polynucleotide sequence of SEQ ID NO:39.

[0055] In some embodiments, exon 2 comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:71; exon 3 comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:72; exon 3a comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:73; exon 4 comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:74; and exon 5 comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:75.

[0056] In other embodiments, exon 2 has a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32; the first part of exon 3 has a polynucleotide sequence of SEQ ID NO:94, and the second part has a polynucleotide sequence of SEQ ID NO:9. Exon 3a has a polynucleotide sequence of SEQ ID NO:12; exon 4 has a polynucleotide sequence of SEQ ID NO:15; exon 4b has a polynucleotide sequence of SEQ ID NO:14; and exon 5 has a polynucleotide sequence of SEQ ID NO:17.

[0057] In other embodiments, exon 2 has a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32; exon 3 has a polynucleotide sequence of SEQ ID NO:34 or SEQ ID NO:56; exon 3a has a polynucleotide sequence of SEQ ID NO:12; exon 4 has a polynucleotide sequence of SEQ ID NO:15; exon 4b has a polynucleotide sequence of SEQ ID NO:14; and exon 5 has a polynucleotide sequence of SEQ ID NO:17.

[0058] In other embodiments, exon 2 has a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32; exon 3 has a polynucleotide sequence of SEQ ID NO:34 or SEQ ID NO:56; exon 3a has a polynucleotide sequence of SEQ ID NO:12; exon 4 has a polynucleotide sequence of SEQ ID NO:15; exon 4b has a polynucleotide sequence of SEQ ID NO:14; exon 5 has a polynucleotide sequence of SEQ ID NO:17; intron 2 has a polynucleotide sequence of SEQ ID NO:55; intron 3 has a polynucleotide sequence of SEQ ID NO:58; and intron 4 has a polynucleotide sequence of SEQ ID NO:39.

[0059] In arthropod female-specific gene expression systems, the promoter can be the Hsp70 promoter, β-tubulin promoter, Hsp83 promoter, protamine promoter, actin promoter, Hsp70 minipromoter, P minipromoter, CMV minipromoter, Acf5C-based minipromoter, TRE3G promoter, BmA3 promoter fragment, or Adh core promoter. In some embodiments, the promoter is the Hsp70 minipromoter (dmHsp70 minipro) derived from Drosophila melanogaster. In other embodiments, the promoter is the human CMV minipromoter (hCMV minipro). In some embodiments, the hCMV minipro also comprises the 5'UTR of turnip yellow mosaic virus (TYMV). In some embodiments, the promoter has the polynucleotide sequence of SEQ ID NO:18, SEQ ID NO:41, SEQ ID NO:63, or SEQ ID NO:65.

[0060] The arthropod female-specific gene expression system of the present invention may further include a transcriptional control element that controls transcription by the presence or absence of a chemical ligand. In some embodiments, the transcriptional control element is a tetracycline-responsive element, and the chemical ligand is tetracycline or an analogue or derivative thereof. In some embodiments, the tetracycline-responsive element is tetOx1, tetOx2, tetOx3, tetOx4, tetOx5, tetOx6, tetOx7, tetOx8, tetOx9, tetOx10, tetOx11, tetOx12, tetOx13, tetOx14, tetOx15, tetOx16, tetOx17, tetOx18, tetOx19, tetOx20, or tetOx21.

[0061] In some embodiments, the arthropod is an insect. In some embodiments, the insect is a noctuid moth. Examples of genera of noctuid moths include, but are not limited to, *Spodoptera*, *Helicoverpa*, *Chrysodeixis*, *Anticarsia*, *Peridroma*, or *Heliothis*. In some embodiments, the insect is the fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), African cotton leafworm (Spodoptera littoralis), bollworm (Helicoverpa armigera), leaf-eating moth (Peridroma saucia), grain moth (Helicoverpa zea), soybean moth (Chrysodeixis includens), bean moth (Anticarsia gemmatalis), or tobacco moth (Heliothis virescens).

[0062] In some implementations, the dsx gene of the Noctuidae family is derived from species of the genera *Spodoptera*, *Helicoverpa*, *Chrysodeixis*, *Anticarsia*, *Peridroma*, or *Heliothis*.

[0063] In some implementations, the dsx gene of the Noctuidae family is derived from the fall armyworm (Spodopterafrugiperda), beet armyworm (Spodoptera exigua), gray-winged armyworm (Spodoptera littoralis), cotton bollworm (Helicoverpa armigera), saucia armyworm (Peridroma saucia), cereal armyworm (Helicoverpa zea), silver armyworm (Chrysodeixis includens), pear bean armyworm (Anticarsia gemmatalis), or tobacco armyworm (Heliothis virescens).

[0064] The arthropod female-specific gene expression system may further include a second expression unit containing a second promoter, a second transcriptional control element controlling transcription in the presence or absence of a chemical ligand, and a second polynucleotide encoding a second functional protein, the coding sequence of which is confined between a second start codon and a second stop codon. The second functional protein encodes Hid or a homolog thereof, Reaper (Rpr) or a homolog thereof, Nipp1Dm or a homolog thereof, calmodulin or a homolog thereof, Michelob-X or a homolog thereof, medea or a homolog thereof, a microRNA toxin, or a nuclease; the first functional protein encodes tTAV or a homolog thereof, tTAV2 or a homolog thereof, tTAV3 or a homolog thereof, or tTAF or a homolog thereof. This provides positive feedback, where, in the presence or absence of a chemical ligand, the transcription factor can drive its own expression and the transcription of the second expression unit.

[0065] In some embodiments, the second expression unit includes a second splice control polynucleotide operatively linked to a second polynucleotide encoding a second functional protein (e.g., a transcription factor), which works in conjunction with the spliceosome in arthropods to enable sex-specific mediated splicing of the primary transcript in arthropods. One sex of the arthropod splices the second splice control polynucleotide to produce an open reading frame containing the second polynucleotide encoding the second functional protein, while the other sex splices the second splice control polynucleotide to produce another reading frame, as follows:

[0066] (a) Not framed with the second polynucleotide encoding the second functional protein;

[0067] (b) cleaving the second polynucleotide encoding the second functional protein; or

[0068] (c) This results in one or more stop codons in another reading frame preventing the translation of the second functional protein.

[0069] In some implementations, the second splice control polynucleotide is the same as the first splice control polynucleotide.

[0070] In some embodiments of the arthropod female-specific gene expression system, the system further includes a second promoter operatively linked to a polynucleotide encoding a marker protein. In some embodiments, the marker protein is a fluorescent protein. In a particular embodiment, the fluorescent protein is DsRed2.

[0071] This invention provides plasmids for preparing genetically engineered insects of the family Noctuidae. In certain embodiments, these plasmids comprise SEQ ID NO:86 (pOX5403), SEQ ID NO:87 (pOX5368), and SEQ ID NO:88 (pOX5382).

[0072] The present invention also provides a method for suppressing wild arthropod (e.g., noctuid moths) populations by releasing genetically engineered male arthropods (e.g., noctuid moths) comprising the expression system of the present invention into a population of wild arthropods of the same species. The genetically engineered arthropods are then mated with the wild arthropods, and the offspring of such mating are differentially spliced ​​with primary transcripts of splice cassettes to produce functional proteins with lethal, harmful, or sterile effects (in the case of female arthropods), resulting in the death of female offspring or the inability of female offspring to reproduce effectively, thereby suppressing the wild arthropod population.

[0073] The present invention also provides a method for reducing, suppressing, or eliminating crop damage caused by arthropods (e.g., noctuid moths), comprising releasing genetically engineered male arthropods (e.g., noctuid moths) containing the expression system of the present invention into a wild arthropod population of the same species, then mating the genetically engineered arthropods with the wild arthropods, and the offspring of such mating selectively splicing primary transcripts of splice cassettes to produce (in the case of female arthropods) functional proteins with lethal, harmful, or sterile effects, causing the death of female offspring or the inability of female offspring to reproduce effectively, thereby suppressing the wild arthropod population and reducing, suppressing, or eliminating crop damage caused by wild insects. Attached Figure Description

[0074] Figure 1 The gene map of the pOX5403 plasmid is shown. The piggyBac 5' and 3' segments are the transposon elements required for insertion of the OX5403 rDNA into the genome of the fall armyworm (Spodoptera frugiperda). The DNA sequence between and including the two piggyBac elements is the rDNA still integrated into the OX5403A genome.

[0075] Figure 2 A linear plasmid diagram is shown, illustrating the two genes (DsRed2, Sfdsx_tTAV) inserted into OX5403A. Due to the splicing module, the tTAV protein is expressed only in females where tetracycline family antibiotics are absent.

[0076] Figure 3Splice variants of the self-limiting tTAV gene are shown. The Sfdsx splicing module consists of Sfdsx exons 2, 3, 3a, 4b, 4, and 5, and Sfdsx introns 2, 3, and 4. In females, female-specific transcripts F1 and F2 are produced. In the absence of a tetracycline antidote, the F1 and F2 transcripts produced in female fall armyworms express the transgene, thereby expressing the tTAV protein in a female-specific manner. The F1 and F2 transcripts contain the Sfdsx start codon fused with ubiquitin_tTAV and the P10 3'UTR. tTAV is within the frame with the start codon, so the F1 and F2 transcripts can be translated into the tTAV protein. In males, only transcript M is produced. Transcript M contains Sfdsx exons 2 and 5, ubiquitin_tTAV, and the P10 3'UTR. Like the other two transcripts, the ORF in this transcript begins upstream of exon 2 of Sfdsx and ends at exon 5 (in a different box than the ORF encoding the tTAV protein). In the M transcript, excluding exons 3, 3a, 4b, and 4 of dsx prevents the production of the tTAV protein because the tTAV coding sequence is not in the box with the tTAV start codon, and is also in the box with the stop codon at the end of exon 5. Arrows indicate the position of the start codon, and red octagons indicate the position of the stop codon within the box. The male transcript is likely to be degraded due to nonsense-mediated decay (Hansen, KD et al. (2009) PLoS Genet. 5, e1000525).

[0077] Figure 4 The gene map of the pOX5368 plasmid is shown. The piggyBac 5' and 3' segments are the transposon elements required for insertion of the OX5368 rDNA into the genome of the fall armyworm (Spodoptera frugiperda). The DNA sequence between and including the two piggyBac elements is the rDNA still integrated into the OX5368 genome.

[0078] Figure 5 A linear plasmid diagram is shown, illustrating the two genes (DsRed2, Sfdsx_tTAV2) inserted into OX5368. Due to the splicing module, the tTAV2 protein is expressed only in females in the absence of tetracycline family antibiotics.

[0079] Figure 6The splice variants of the self-limiting tTAV gene are shown. The Sfdsx splicing module consists of Sfdsx exons 2, 3, 3a, 4b, 4, and 5, and Sfdsx introns 2, 3, and 4. In females, female-specific transcripts F1 and F2 are produced. The F1 and F2 transcripts produced in female fall armyworms express the transgene in the absence of a tetracycline antidote, resulting in female-specific expression of the tTAV protein. The F1 and F2 transcripts contain the tTAV coding sequence and the DmK10 3'UTR, thus enabling translation into the tTAV protein. In males, only transcript M is produced. Transcript M contains Sfdsx exons 2 and 5, and the DmK10 3'UTR. This transcript does not encode the tTAV protein, producing only a short fragment of Sfdsx. Arrows indicate the position of the start codon, and red octagons indicate the position of the stop codon within the box. The sequences of these transcripts and their predicted encoded proteins are given in Appendix 5. Male transcripts are likely degraded by nonsense-mediated decay (Hansen et al., 2009).

[0080] Figure 7 The gene map of the pOX5382 plasmid is shown. The piggyBac 5' and 3' are the transposon element sequences required for insertion of the OX5382 rDNA into the fall armyworm genome. The DNA sequence between and including the two piggyBac elements is the rDNA still integrated into the OX5382G genome.

[0081] Figure 8 A linear plasmid diagram is shown, illustrating the two genes (DsRed2, Sfdsx_tTAV) inserted into OX5382G. Due to the splicing module, the tTAV protein is expressed only in females where tetracycline family antibiotics are absent.

[0082] Figure 9Splice variants of the self-limiting tTAV gene are shown. The Sfdsx splicing module consists of Sfdsx exons 2, 3, 3a, 4b, 4, and 5, and Sfdsx introns 2, 3, and 4. In females, female-specific transcripts F1 and F2 are produced. In the absence of a tetracycline antidote, the F1 and F2 transcripts produced by the female fall armyworm express the transgene, thereby expressing the tTAV protein in a female-specific manner. The F1 and F2 transcripts contain the Sfdsx start codon fused with ubiquitin_tTAV and the P10 3'UTR. tTAV is within the frame with the start codon, so the F1 and F2 transcripts can be translated into the tTAV protein. In males, only transcript M is produced. Transcript M contains Sfdsx exons 2 and 5, ubiquitin_tTAV, and the P10 3'UTR. Like the other two transcripts, the ORF in this transcript begins upstream of exon 2 of Sfdsx and terminates at exon 5 (in a different box than the ORF encoding the tTAV protein). In the M transcript, the exclusion of exons 3, 3a, 4b, and 4 of dsx prevents the production of the tTAV protein because the tTAV coding sequence is outside the box with the tTAV start codon and inside the box with the stop codon located at the end of exon 5. Arrows indicate the position of the start codon, and red octagons indicate the position of the stop codon inside the box. The sequences of these transcripts and their predicted encoded proteins are given in Appendix 5. The male transcript may be degraded due to nonsense-mediated decay (Hansen et al., 2009).

[0083] Figure 10 The results of breeding hemizygous noctuid moths with or without tetracycline during larval feeding are shown; the shadow moth contains a female-specific gene expression system, while the white moth is wild-type; when raised with tetracycline, the female-specific expression system is shut off, and both male and female offspring can survive to adulthood; when raised without tetracycline, a copy of the female-specific gene expression system may be inherited by offspring, and in moths that inherit the female-specific gene expression system, only males survive to adulthood.

[0084] Figure 11 The survival rates of males and females of OX5368C are shown in the presence and absence of doxycycline. No females survived in the absence of doxycycline.

[0085] Figure 12 The survival rates of males and females of OX5403A are shown in the presence and absence of doxycycline. No females survived in the absence of doxycycline.

[0086] Figure 13 The survival rates of males and females of OX5382G are shown in the presence and absence of doxycycline. No females survived in the absence of doxycycline.

[0087] Figure 14 The survival rates of males and females of OX5382J are shown in the presence and absence of doxycycline. No females survived in the absence of doxycycline.

[0088] Figure 15 The DsRed2 fluorescence of the OX5382B transgenic fall armyworm at various life stages is shown compared to the wild-type fall armyworm.

[0089] Figure 16 Splicing patterns of selected dsx exons 2, 3, 3a, 4b, 4, and 5 of the Noctuidae family are shown: A: Splicing patterns of female (top) and male (bottom) cotton bollworms (black box, exons; gray box, variable splicing sites within exons; white box: 3'UTR-type sequence; *: stop codon), as Wang XY et al. (2014) Insect Biochem. Mol. Biol. 44: 1-11; B: Splicing patterns of female (top) and male (bottom) fall armyworms (black box, exons; gray box, variable splicing sites within exons); C: Details of endogenously spliced ​​female (F1, F2, F3, and F4) and male transcripts (stop symbols indicate stop codons).

[0090] Figure 17 The amino acid sequences of exons 2, 3, 3a, 4, and 5 encoded by the female (F) and male (M) transcripts of dsx, constructed from OX5403, OX5368, OX5382, endogenous wild-type fall armyworm (Endo) and cotton bollworm (HA), are shown. A: Exon 2 of the male and female transcripts; B: Exon 3 (used only for the female transcript); C: Exon 3a of the female transcripts from OX5403 and OX5382; D: Exon 4 of the female transcripts from OX5403 and OX5382; E: Exon 5 of the female transcripts from OX5403 and OX5382; F: Exon 5 of the male transcript; the shaded areas in HA represent conserved amino acids in Lepidoptera (Wang). XY et al. (2014); OX5403, OX5368, OX5382, shaded areas of wild-type fall armyworm indicate amino acid identity with conserved amino acids in cotton bollworm.

[0091] Figure 18An implementation of a female-specific expression system is shown, which contains only exons 2, 3, and 5 as part of a splice cassette; in females, splicing results in the binding of exons 2, 3, and 5 (in this case, within the cassette along with the ubiquitin leader sequence and the tTAV gene), leading to female death. In males, splicing results in the joining of exons 2 and 5, such that the stop codon is translated before the ubiquitin leader sequence or the tTAV sequence, thus allowing male survival.

[0092] Figure 19 One embodiment is shown in which a lethal, harmful, or sterile gene (tTAV in this case) is located between the dividing exons 3, which are connected to the 5' and 3' portions of exon 3 via adapters. In a specific example, the first portion of exon 3 (exon 3p1; SEQ ID NO: 94) is connected to the tTAV open reading frame (ORF; SEQ ID NO: 99) via an adapter (adapter 1; SEQ ID NO: 95), and sequentially connected to the second portion of exon 3 (exon 3p2; SEQ ID NO: 9) via a second adapter (adapter 2; SEQ ID NO: 96). Invention Details

[0093] This specification contains citations of various journal articles, patent applications, and patents. These are incorporated into the text as if each item were fully described within the text.

[0094] As used herein, the term "exon" refers to the full-length exon of dsx and portions thereof for ease of reference. Thus, "exon 2 of dsx" refers to the full-length wild-type dsx exon 2 as well as the truncated form of exon 2. "Exons" also include full-length or truncated exons containing point mutations that remove the putative internal start codon (atg) or stop codon, thus potentially preserving or losing open reading frames. The 5' and 3' boundaries of exons / introns must preserve splicing donor and acceptor sites to allow splicing of one exon to another. In some cases, the specification will refer to "truncated exon" to indicate that portions of the wild-type exon have been deleted. In other cases, this specification will refer to "modified exon," referring to an exon into which certain mutations have been introduced, modifying the polynucleotide sequence in the wild-type dsx exon sequence. Specific embodiments of exons are also referenced to their respective SEQ ID NOs. Likewise, it should be understood that an exon refers to a polynucleotide sequence that can be translated into different reading frames to produce different polypeptide sequences. A concrete example is that the construct allows the translation of exon 5 in certain female constructs to produce the amino acid sequence of SEQ ID NO: 89, while in males, the polynucleotide sequence is read in a different reading frame to produce the amino acid sequence of SEQ ID NO: 78.

[0095] The term "intron" refers to a polynucleotide sequence that is part of the primary transcript of an RNA molecule, but is cut off from the final RNA to be translated.

[0096] As used herein, the term "penetration" refers to the proportion of individuals carrying a specific variant of a gene that also expresses a phenotypic trait associated with that variant. Therefore, in relation to this invention, "penetration" refers to the proportion of transformed organisms expressing a lethal phenotype.

[0097] As used herein, the term "construct" refers to an artificially constructed segment of DNA used for insertion into a host organism to genetically modify the host organism's DNA. At least a portion of the construct is inserted into the host organism's genome and alters the host organism's phenotype. The construct may form part of a vector or be the vector itself.

[0098] As used herein, the term "transgenic" refers to a polynucleotide sequence containing a first gene expression system and a second gene expression system to be inserted into the genome of a host organism to alter the host organism's phenotype. The portion of a plasmid vector containing the gene to be expressed is referred to herein as transforming DNA or recombinant DNA (rDNA).

[0099] As used herein, the term "gene expression system" refers to the expression of a gene along with any gene and DNA sequence required to express the gene to be expressed.

[0100] As used herein, the term "splicing control sequence" refers to a gene-associated RNA sequence, which, together with the spliceosome, mediates the alternative splicing of the RNA product of the gene. Preferably, the splicing control sequence, together with the spliceosome, mediates the splicing of the RNA transcript of the associated gene to produce mRNA encoding a functional protein, and mediates alternative splicing of the RNA transcript to produce at least one alternative mRNA encoding a non-functional protein. A "splicing control module" may comprise multiple splicing control sequences that link multiple exons to form a nucleic acid encoding a polypeptide.

[0101] As used herein, the term "trans-activation activity" refers to the activity of an activating transcription factor that leads to increased gene expression. An activating transcription factor may bind to a promoter or operator gene operatively linked to the gene, thereby activating the promoter and thus enhancing gene expression. Alternatively, an activating transcription factor may bind to an enhancer associated with the promoter, thereby promoting the activity of the promoter through the enhancer.

[0102] As used in this article, the term "lethal gene" refers to a gene whose expression product, in sufficient quantities, is lethal to the organism expressing the lethal gene.

[0103] As used herein, the term "lethal effect" refers to a harmful or sterile effect, such as the ability to kill an organism itself or its offspring, or the ability to reduce or destroy the function of certain tissues, particularly reproductive tissues, thereby rendering the organism or its offspring sterile. Thus, some lethal effects (e.g., poisons) will kill an organism or tissue within a short timeframe relative to its lifespan, while others may simply impair the organism's function, such as reproductive function.

[0104] As used herein, the term "tTAV gene variant" refers to a polynucleotide that encodes a functional tTA protein but with a different nucleotide sequence. These nucleotides can encode different tTA protein sequences, such as tTAV2 and tTAV3, for example, SEQ ID NO:97 and SEQ ID NO:98, respectively.

[0105] As used herein, the term "promoter" refers to a DNA sequence typically located directly upstream of the coding sequence required for basic transcription and / or regulatory transcription of a gene. In particular, a promoter is sufficient to allow the initiation of transcription and typically has a transcription initiation site and a binding site for the RNA polymerase transcription complex.

[0106] As used herein, the term "minimal promoter" refers to a promoter as defined above, which typically has a transcription initiation site and a polymerase complex binding site, and further typically has sufficient additional sequences to allow both to function effectively. Other sequences, such as tissue-specific sequences, may be missing.

[0107] As used herein, the term "exogenous control factor" refers to a substance that is not naturally present in the host organism, is not present in the host organism's natural habitat, or is not present in the environmental conditions of the host organism's natural habitat. Therefore, the presence of exogenous control factors is controlled by the manipulator of the transformed host organism in order to control the expression of the gene expression system.

[0108] As used herein, the term "tetO element" refers to one or more tandemly located tetO operator gene units. As used herein, the term "tetOx (number)" refers to a tetO element consisting of an indicated number of tetO operator gene units. Thus, a reference to "tetOx7" indicates a tetO element consisting of 7 tetO operator gene units. Similarly, a reference to "tetOx14" refers to a tetO element consisting of 14 tetO operator gene units, and so on.

[0109] When referring to a specific nucleotide or protein sequence, it should be understood that this includes referring to any mutant or variant that has substantially equivalent biological activity. Preferably, the mutant or variant has at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 99%, preferably at least 99.9%, and most preferably at least 99.99% sequence identity with the reference sequence.

[0110] However, it will be understood that despite the aforementioned sequence homology, certain elements, particularly flanking nucleotides and splice branching sites, must be preserved for the system to function effectively. In other words, while some parts may be deleted or altered, at least 30%, preferably 50%, more preferably 70%, more preferably 90%, and most preferably 95% of the alternative splicing function or activity should be preserved compared to the wild type. This can also be increased, for example, by appropriately modifying the binding sites of alternative splicing factors or sites that interact with the spliceosome compared to the wild type.

[0111] As used in this article, "splicing control module" refers to a polynucleotide construct incorporated into a vector that undergoes differential splicing (e.g., stage-specific, sex-specific, tissue-specific, species-specific, etc.) when introduced into insects. Therefore, if the splicing control module performs differential splicing in a sex-specific manner, it produces transcripts that are different in females than in males.

[0112] As used in this article, “5'UTR” refers to the untranslated region of an RNA transcript, which is the 5' translated portion of the transcript and usually contains the promoter sequence.

[0113] As used in this article, “3'UTR” refers to the untranslated region of an RNA transcript, which is the 3' translated portion of the transcript and usually contains polyadenylated sequences.

[0114] This invention provides plasmids, expression constructs, and arthropods, particularly noctuid moths, having elements for sex-specific expression of a lethal gene that causes death in one sex of the noctuid moth. The elements are repressible, for example, by a chemical entity (e.g., tetracycline or an analogue thereof). In certain embodiments, the present invention relates to noctuid insects transformed with these constructs, particularly those belonging to the genera *Spodoptera*, *Helicoverpa*, *Chrysodeixis*, *Anticarsia*, *Peridroma*, or *Heliothis*, including, but not limited to, *Spodopterafrugiperda* (fall armyworm), *Spodoptera exigua* (beef armyworm), *Spodoptera littoralis* (African cotton leafworm), *Helicoverpa armigera* (cotton bollworm; larva; archipelagic moth; African cotton bollworm), *Peridroma saucia* (spotted leafworm), *Helicoverpazea* (larva), and *Chrysodeixis*. Includens (soybean noctuid moth), Anticaria spp. (velvet bean caterpillar), and Heliothis virescens (tobacco noctuid moth larva).

[0115] splicing control module

[0116] This invention provides a splicing control module polynucleotide sequence that enables differential splicing in an organism (e.g., sex-specific, stage-specific, germline-specific, tissue-specific, etc.). In particular, this invention provides a splicing control module that provides sufficient female-specific expression of a useful target gene. In some embodiments of this invention, the target gene is a gene that confers harmful, lethal, or sterile effects. For convenience, this specification will refer to lethal effects; however, it should be understood that the splicing module can be used for other genes of interest, as described in further detail below.

[0117] Because at least one splicing control module is operatively linked to the target gene to be differentially expressed in each gene expression system, the expression of a transgenic dominant lethal gene can be sex-specific, or a combination of sex-specific and stage-specific, germline-specific, or tissue-specific. In some implementations, sex-specific expression is female-specific. In addition to the promoter, the splicing control module in each gene expression sequence allows for control of additional levels of protein expression.

[0118] The gene for the splicing control module contains a protein or polypeptide coding sequence, i.e., at least two or more exons capable of encoding a polypeptide, such as a protein or fragment thereof. Preferably, different exons are differentially spliced ​​together to provide a variable mRNA. Preferably, the alternatively spliced ​​mRNA has different coding potentials, i.e., encoding different protein or polypeptide sequences. Therefore, the expression of the coding sequence is regulated by alternative splicing.

[0119] Each splice control module in the system includes at least one splice acceptor site and at least one splice donor site. The number of donor and acceptor sites can vary depending on the number of sequence segments to be spliced ​​together.

[0120] In some embodiments, the splicing control module regulates alternative splicing through both intron and exon nucleotides. It will be understood that in alternative splicing, the sequence can be intronic in some cases (i.e., in certain alternative splicing variants where the intron is spliced ​​out), and exonic in others. In other embodiments, the splicing control module is an intron splicing control module. In other words, preferably, the splicing control sequence is substantially derived from a polynucleotide that forms part of an intron and is therefore excised from the primary transcript by splicing, such that these nucleotides are not retained in the mature mRNA sequence.

[0121] As mentioned above, exon sequences can participate in mediating alternative splicing, but it is preferred that at least some intron control sequences participate in mediating alternative splicing.

[0122] The splicing control module can be removed from or retained in the mRNA precursor via splicing to encode a fusion protein encoding at least a portion of the target gene to be differentially expressed. Preferably, the splicing control module does not cause a frameshift in the resulting splice variant. Preferably, this is a splice variant encoding a full-length functional protein.

[0123] The interaction between the splicing control module and cellular splicing machinery, such as the spliceosome, results in or mediates the removal of a series of, for example, at least 20, 30, 40, or 50 or more consecutive nucleotides from the primary transcript, which would link (splice) discontinuous nucleotide sequences in the original transcript together (because they, or their complementary sequences (if antisense sequences are considered), are discontinuous in the original template sequence for transcribing the primary transcript). The series of at least 50 consecutive nucleotides contains introns. This mediation preferably operates in a sex-specific, more preferably female-specific, manner, such that equivalent primary transcripts of different sexes, and optionally equivalent primary transcripts at different stages, tissue types, etc., tend to remove introns of different sizes or sequences, or may remove introns in some cases but not in others. This phenomenon, the removal of introns of different sizes or sequences under different conditions, or the differential removal of introns of a given size or sequence under different conditions, is called alternative splicing. Alternative splicing is a well-known phenomenon in nature, and many examples are known.

[0124] When alternative splicing is sex-specific, preferably, the splice variant encoding the functional protein to be expressed in the organism is an F1 splice variant or an F2 splice variant (or both F1 and F2), that is, a splice variant, where F indicates that it is found only or primarily in females, although this is not required.

[0125] When removing exon nucleotides, if you want to avoid frameshift, you must remove them in multiples of three (the entire codon); if you want to introduce frameshift, you remove them in multiples of one or two nucleotides (not multiples of three). It should be understood that removing only one or more multiples of two nucleotides could result in a completely different protein sequence encoding at or near the splice site of the mRNA.

[0126] Accordingly, for configurations in which all or part of the functional open reading frames are located on cascade exons, preferably, the cascade exons include transcripts found only or primarily in females, and preferably, such transcripts are alone or in combination, the most abundant variants found in females, although this is not required.

[0127] In a preferred embodiment, the sequence is contained in a hybrid or recombinant sequence or construct derived from a naturally occurring intron sequence that itself has undergone alternative splicing in its natural or original context. Thus, an intron sequence can be considered as part of an intron formed in at least one alternative splicing variant of a natural analog. Therefore, sequences corresponding to a single, continuous fragment of a naturally occurring intron sequence are contemplated, as are hybrid sequences of such sequences, including hybrid sequences from two different naturally occurring intron sequences, and sequences with deletions or insertions relative to a single, continuous fragment of a naturally occurring intron sequence, and their hybrids. In this invention, the sequence derived from a naturally occurring intron sequence can itself be associated with a sequence that is not itself part of any naturally occurring intron. If such sequences are transcribed and preferably retained in mature RNA in at least one splicing variant, they can be considered exons.

[0128] It should also be understood that the term "frameshift" can also refer to directly encoding a stop codon, which can also lead to nonfunctional proteins, such as the disruption of mRNA sequences caused by nucleotide insertion or deletion. In addition to generating two or more different protein or polynucleotide sequences with one or more unpredictable or identifiable functions, the generation of different splice variants of two or more different protein or polypeptide sequences with different functions is also envisioned. Furthermore, the generation of different splice variants of two or more different protein or polypeptide sequences with similar functions but different subcellular locations, stability, or ability to bind or associate with other proteins or nucleic acids is also envisioned.

[0129] The modified dsx intron is one example. In this case, as done in the embodiments, it is preferable to delete an appropriate number of introns from the alternatively spliced ​​introns, for example, 90% or more in some cases, while still retaining the functionality of alternative splicing. Therefore, while large deletions are contemplated, smaller deletions, such as even single nucleotide insertions, substitutions, or deletions, are also contemplated as preferred.

[0130] Splice module bisexuality (dsx)

[0131] Introns are typically composed of the following features (referred to here as sense DNA sequences 5' to 3'); in RNA, thymine (T) will be replaced by uracil (U):

[0132] a. 5' end (referred to as the splice "donor"): GT (or possibly GC)

[0133] b. 3' end (called the splice "receptor"): AG

[0134] c. Upstream of the receptor / 5' (called the "branch point"): A-polypyrimidine bundle, i.e., AYYYYY…Y n

[0135] The terminal nucleotide of the exon immediately adjacent to the 5' intron splicing "donor" and the 3' intron splicing "acceptor" is usually G.

[0136] In some implementations, the splice control module is positioned immediately adjacent to the start codon in the 3' direction, such that the G of the ATG is the 5' of the start (5' end) of the splice control module. This can be advantageous because it allows the G of the ATG start codon to be a 5'G flanking sequence of the splice control module.

[0137] Alternatively, the splicing control module is located at 3' of the start codon, but within 10,000 exon bp, 9,000 exon bp, 8,000 exon bp, 7,000 exon bp, 6,000 exon bp, 5,000 exon bp, 4,000 exon bp, exon 3,000 bp, exon 2,000 bp or 1,000 exon bp, 500 exon bp, 300 exon bp, 200 exon bp, 150 exon bp, 100 exon bp, 75 exon bp, 50 exon bp, 30 exon bp, 20 exon bp or 10 or even 5, 4, 3, 2 or 1 exon bp.

[0138] Preferably, as described above, the branch point is included in each splice control sequence. The branch point is the sequence in which the splice donor is initially connected, indicating that splicing occurs in two stages, in which the 5' exon is separated and then connected to the 3' exon.

[0139] The provided sequence is resistant to certain sequence variations and will still splice correctly. Several important nucleotides are known. These are essential for all splicing. The initial GU and final AG of the introns are particularly important and therefore preferred, as discussed elsewhere, although approximately 5% of introns begin with GC. This common sequence is preferred, although it works for all splicing but not particularly for alternative splicing.

[0140] In insects, the dsx gene consists of introns and exons, which are differentially spliced ​​between males and females. The splice cassette of this invention is derived from the insect dsx gene and can be derived from any insect source, as long as the primary transcript is differentially spliced ​​between males and females. In some embodiments, the insect dsx sequence is derived from species of the genera *Spodoptera*, *Helicoverpa*, *Chrysodeixis*, *Anticarsia*, *Peridroma*, or *Heliothis*. In specific instances, the dsx gene is derived from the following species of the Noctuidae family, including, but not limited to, *Spodoptera frugiperda*, *Spodoptera exigua*, *Spodopteralittoralis*, *Helicoverpa armigera*, *Peridroma saucia*, *Helicoverpa zea*, *Chrysodeixis includens*, *Anticarsiagemmatalis*, or *Heliothis virescens*. In a particular instance, dsx is derived from *Spodoptera frugiperda*.

[0141] The dsx splice cassette of the present invention includes introns and exons, enabling differential splicing. In some embodiments, the splice cassette includes at least exon 2, intron 2, exon 3, intron 4, and exon 5 of dsx. In such embodiments, a lethal gene (e.g., tTAV or a variant thereof) can be operatively linked to the middle of exon 2, the 3' of intron 2, and exon 3, but not the 5' of intron 4 and exon 5 (see [link to previous embodiment]). Figure 6 and Figure 19 Therefore, the female will splice the product of exon 2-exon 3-tTAV-exon 4-exon 5, and the male will cleave off the tTAV to provide exon 2-exon 5 (see example). Figure 6 This construct may also contain exons 3a, 4, 4b and intron 3.

[0142] In other arrangements, the lethal gene (e.g., tTAV) can be the 3' of exon 2, intron 2, exon 3, exon 3a, intron 3, exon 4b, exon 4, intron 4, and exon 5 of the dsx splicing module element. In such an embodiment, the female splices the primary transcript of the splicing module to produce exon 2-exon 3-exon 4-exon 5 (see, for example, SEQ ID NO:76) or exon 2-exon 3-exon 3a-exon 4-exon 5 (see, for example, SEQ ID NO:77), while the male splices the primary transcript of the splicing cassette to produce exon 2-exon 5, wherein a stop codon is present before translation of the lethal protein (see...). Figure 3 and Figure 9 For example, such a stop codon might be due to splicing exon 2 to exon 5, where exon 5 and exon 2 are not within the frame.

[0143] In some embodiments, exon 2 has a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:71. Exon 2 may have, for example, a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32. In some embodiments, exon 3 has a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:72. Exon 3 may have, for example, a polynucleotide sequence of SEQ ID NO:94, SEQ ID NO:34, or SEQ ID NO:56. In some embodiments, exon 3a has a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:73. Exon 3a may have, for example, a polynucleotide sequence of SEQ ID NO:12. In some embodiments, exon 4 has a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:74. Exon 4 may have, for example, a polynucleotide sequence of SEQ ID NO:15. In some embodiments, exon 5 has a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:75. Exon 5 may have, for example, a polynucleotide sequence of SEQ ID NO:17.

[0144] Exon 4b binds to exon 4 in the absence of intercalated introns. Instead, there appears to be an internal splicing recognition site that allows Noctuidae to cleave exon 4b from the primary transcript, leaving exon 4 intact. Therefore, exon 4b / exon 4 can be incorporated into constructs such as SEQ ID NO:90 ( Figure 3 ), SEQ ID NO:91 ( Figure 6 ) or SEQ ID NO:92( Figure 9 The construct shown in the diagram can be used, or a construct without exon 4b can be used.

[0145] In some embodiments, intron 2 has the polynucleotide sequence of SEQ ID NO:55. In some embodiments, intron 3 has the polynucleotide sequence of SEQ ID NO:58. In some embodiments, intron 4 has the polynucleotide sequence of SEQ ID NO:39. The length of the introns can vary, as long as the splice donor and splice acceptor sites are conserved. The specific intron sequences provided herein and in the examples are merely exemplary, and those skilled in the art will know how to modify the sequence and length of such introns to allow proper splicing from the primary transcript to the exons.

[0146] This article provides examples of complete splicing control modules for SEQ ID NO:6, SEQ ID NO:31, and SEQ ID NO:53.

[0147] Target heterologous genes

[0148] This system is capable of expressing at least one target protein, i.e., a functional protein expressed in an organism. Such a target protein may have a therapeutic effect or may be a marker, such as a fluorescent protein (e.g., AmCyan, Clavularia, ZsGreen, ZsYellow, Discosoma striata, DsRed2, AsRed, Discosoma Green, Discosoma Magenta, HcRed-2A, mCherry, green fluorescent protein (GFP), red fluorescent protein (RFP), and HcRed-Cr1-tandem, or one or more mutants or variants thereof), or other markers well known in the art, such as drug resistance genes. Other target proteins may be, for example, proteins with harmful, lethal, or infertile effects. Alternatively, the target heterologous gene may encode an RNA molecule with repressive effects. Other proteins expressed in the organism are envisioned to be combined with the said functional protein, preferably a lethal gene as described below.

[0149] Preferably, in an organism, expression of the heterologous polynucleotide sequence produces phenotypic results. In some embodiments, the functional protein is not a β-galactosidase, but may be associated with visible markers (including fluorescence), survival rate, fertility, reproductive capacity, adaptability, flight ability, vision, and behavioral differences. Of course, it should be understood that in some embodiments, the expression system is often conditional, and the phenotype is expressed only under certain conditions, such as under restrictive or permissive conditions.

[0150] Heterologous polynucleotide sequences can be expressed in Noctuidae. By "heterologous," it should be understood that this refers to a sequence that would not normally associate or connect with at least one element or component of at least one splice control sequence in the wild type. For example, when the splice control sequence originates from a particular organism and the heterologous polynucleotide is a coding sequence for a protein or polypeptide—that is, a polynucleotide sequence encoding a functional protein—the coding sequence can be partially or wholly derived from a gene of the same organism, provided that at least some portions of the transcribed polynucleotide sequence originate from a different source than at least one splice control sequence. Alternatively, the coding sequence can originate from different organisms, and in this case, it can be considered "exogenous." Heterologous polynucleotides can also be considered "recombinant" because the coding sequence for the protein or polypeptide originates from a different location within the same genome (i.e., the genome of a single species or subspecies), or from a different genome (i.e., from the genome of a different species or subspecies) or synthetic origin.

[0151] Heterologous can refer to sequences other than splicing control sequences, and thus can relate to the fact that promoters and other sequences, such as 5'UTR and / or 3'UTR, can be heterologous to the polynucleotide sequence to be expressed in an organism, provided that in the wild type, i.e., in the natural background (if any) of the polynucleotide sequence, the polynucleotide sequence is not found to be associated with or operatively linked to the promoter, 5'UTR, and / or 3'UTR.

[0152] It should be understood that heterologous also applies to “designer” or heterozygous sequences that are not derived from a specific organism but are based on many components from different organisms, as this would also satisfy the requirement that the sequence and at least one component of the splice control sequence are not connected or found to be associated in the wild type, even if a portion or element of the heterozygous sequence is found, provided that at least a portion or element is not. It should also be understood that synthetic forms of naturally occurring sequences can be conceived. Such synthetic sequences are also considered heterologous unless they have the same sequence as sequences that are typically found in the wild type or in the natural environment to be associated with or connected to at least one element or component of at least one splice control sequence.

[0153] This also applies to cases where the heteropolynucleotide is a polynucleotide used to interfere with RNA.

[0154] In one embodiment, when the polynucleotide sequence to be expressed contains a coding sequence for a protein or polypeptide, it should be understood that reference to expression in an organism refers to providing one or more transcribed RNA sequences, preferably mature mRNA. However, preferably, this may also refer to a translational polypeptide in the organism.

[0155] Lethal genes

[0156] In some embodiments, the functional protein expressed in the organism has lethal or harmful effects. When lethal effects are mentioned herein, it should be understood that this extends to harmful or sterile effects, such as those capable of killing the organism itself or its offspring, or those capable of reducing or disrupting the function of certain tissues, with reproductive tissues being particularly preferred, thus rendering the organism or its offspring sterile. In other embodiments, systems that are not lethal but harmful may be employed, thereby imposing considerable adaptive costs on the organism. Non-limiting examples include blindness and inability to fly (for organisms that are normally capable of flight). Thus, some lethal effects (e.g., poisons) will kill the organism or tissues within a short period of time relative to its lifespan, while others may simply impair the organism's function, such as reproductive function.

[0157] In some implementations, the lethal effect results in sterility, enabling the organism to compete with wild organisms in its natural environment (“in the wild”), but the sterile organism subsequently cannot produce surviving offspring. In this way, the present invention achieves similar or better results than techniques such as insect sterility techniques (SIT) in insects, without the problems associated with SIT, such as cost, danger to users, reduced competitiveness of irradiated organisms, and lack of available and practical sex identification systems.

[0158] In some embodiments, the system includes at least one positive feedback mechanism, namely, differential expression of at least one functional protein via alternative splicing, and at least one promoter, wherein the gene product to be expressed serves as a positive transcriptional control factor for said at least one promoter, thereby making the expression of the product or product controllable. In some embodiments, an enhancer is associated with the promoter, and the gene product is used to enhance the activity of the promoter through the enhancer.

[0159] This invention allows for selective control of the expression of dominant lethal genes, thereby providing selective control over the expression of lethal phenotypes. Therefore, it should be understood that each lethal gene encodes a functional protein, such as Hid, Reaper (Rpr), Nipp1Dm, calmodulin, Michelob-X, tTAV, tTAV2, tTAV3, tTAF, and other tetracycline systems, the Barnase / Barstar combination, medea microRNA toxins, and nucleases, such as, but not limited to, FokI or EcoRI.

[0160] Each lethal gene possesses conditional lethality. Examples of suitable conditions include temperature, such that lethality is expressed at one temperature and not expressed or expressed to a lesser extent at another. Another example of suitable conditions is the presence or absence of a substance, thereby expressing lethality in the presence or absence of the substance, but not simultaneously in both. Preferably, the action of a lethal gene is conditional, and it is not expressed under permissible conditions requiring the presence of a substance absent in the organism's natural environment, thus the lethality of the lethal system occurs in the organism's natural environment.

[0161] Each lethal genetic system can act on a specific cell or tissue or exert its effects on the entire organism. Systems that are not strictly lethal but incur significant adaptive costs are also conceived, such as causing blindness, inability to fly (for organisms that can normally fly), or infertility. Systems that interfere with sex determination are also envisioned, such as converting all or part of an organism from one sex type or tending to convert it to another.

[0162] In some embodiments, the product of at least one lethal gene is preferably an apoptosis-inducing factor, such as the AIF protein or a homolog described in Candé et al. (2002) J. Cell Science 115: 4727-4734. AIF homologs have been found in mammals and even in invertebrates (including insects, nematodes, fungi, and plants), meaning that AIF genes are conserved throughout the eukaryotic world. In other embodiments, the product of at least one lethal gene is Hid, a protein product of the head degeneration defect gene in Drosophila melanogaster, or Reaper (Rpr), a product of the reaper gene in Drosophila, or a mutant thereof. Heinrich and Scott (2000) Proc. Natl. Acad. Sci. USA 97: 8229-8232 describes the use of Hid. Horn and Wimmer (2003) Nature Biotechnology 21: 64-70 describes the use of the mutant derivative HidAla5. White et al. (1996); Science 271(5250):805-807; Wing et al. (2001) Mech.Dev. 102(1-2):193-203; and Olson et al. (2003) J. Biol. Chem. 278(45):44758-44768 describe the use of the mutant derivative RprKR of Rpr. Rpr and Hid are both pro-apoptotic proteins and are thought to bind to IAP1. IAP1 is a highly conserved anti-apoptotic protein. Therefore, even though their sequences are not very conserved, Rpr and Hid are expected to play a role in a wide range of phylogenetic processes (Huang et al. (2002); Vernooy et al. (2000) J. Cell Biol. 150(2):F69-76).

[0163] In some embodiments, Nipp1Dm, a Drosophila homologue of the mammalian Nipp1, is used (Parker et al. (2002) Biochemical Journal 368: 789-797; Bennett et al. (2003) Genetics 164: 235-245). As those skilled in the art will understand, Nipp1Dm is another example of a lethal protein if expressed at appropriate levels. In fact, many other examples of lethal proteins are known to those skilled in the art.

[0164] In other embodiments, the lethal gene is a variant of the tTA, tTAV, or tTAF gene, where tTA stands for "tetracycline repressive transactivator" and V stands for "variant." tTAV is an analogue of tTA, wherein the sequence of tTA is modified to enhance compatibility with the desired insect species. tTAV variants encoding the tTA protein are possible, such that the tTAV gene product has the same function as the tTA gene product. Therefore, variants of the tTAV gene contain modified nucleotide sequences compared to the tTA nucleotide sequence and each other, but encode a protein with the same function. Therefore, tTAV gene variants can be used instead of tTA. Examples of usable tTAVs and their variants include, but are not limited to, tTAV (SEQ ID NO:10), tTAV2 (SEQ ID NO:67), and tTAV3 (SEQ ID NO:68, which encode proteins of SEQ ID NO:80, SEQ ID NO:97, and SEQ ID NO:98, respectively). In some embodiments, the tTA variant proteins contain amino acid substitutions, additions, or deletions. Any combination of lethal genes can be used, and in some embodiments, the lethal genes are the same, while in others, they are different. By accumulating lethal products, increased penetrance of lethal effects and earlier onset of lethality can be achieved.

[0165] In some implementations, the lethal gene causes at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% mortality in insects.

[0166] In some embodiments, if it is desired that more than one feedback loop has more than one lethal gene, each of the first and second lethal genes can independently be a variant of the tTA or tTAV gene. In some embodiments, each of the first and second lethal genes is independently a gene encoding tTAV (SEQ ID NO: 80), tTAV2 (SEQ ID NO: 97), and tTAV3 (SEQ ID NO: 98). In other embodiments, the first and second lethal genes are the same. In a further embodiment, one of the first and second lethal genes encodes tTAV (SEQ ID NO: 80), and the other gene encodes tTAV3 (SEQ ID NO: 68). However, any combination of tTAV variants can be used. Thus, in some embodiments, one of the first and second genes encodes tTAV (SEQ ID NO: 80), and the other encodes tTAV2 (SEQ ID NO: 97), while in another embodiment, one of the first and second genes encodes tTAV2 (SEQ ID NO: 97), and the other gene encodes tTAV3 (SEQ ID NO: 98). In other embodiments, the first lethal gene encodes tTAV (SEQ ID NO:80), and the second lethal gene encodes tTAV3 (SEQ ID NO:98). Examples of polynucleotides encoding tTAV, tTAV2, and tTAV3 are provided as SEQ ID NO:10, SEQ ID NO:81, and SEQ ID NO:82, respectively.

[0167] The polynucleotide sequence to be expressed with lethal, harmful, or infertile effects may contain a polynucleotide for interfering RNA (RNAi). In some embodiments, when the polynucleotide sequence to be expressed contains a polynucleotide for interfering RNA, it should also be understood that reference to expression in an organism refers to the interaction of the polynucleotide for interfering RNA or its transcript in the RNAi pathway. This can be achieved, for example, by binding a digestion enzyme (Dicer) (an enzyme similar to RNA Pol III) or by forming small interfering RNA (siRNA). Such sequences can provide, for example, one or more segments of double-stranded RNA (dsRNA), preferably in the form of a primary transcript, which can be processed by a digestion enzyme (Dicer). Such segments include, for example, single-stranded RNA segments that can form loops, such as segments found in short hairpin RNA (shRNA), or longer regions that are substantially self-complementary.

[0168] Particularly in insects and nematodes, it is preferable to provide a portion of dsRNA, for example, via hairpin formation, which can then be processed by a digestion enzyme (Dicer) system. Mammalian cells typically produce an interferon response to long dsRNA sequences; therefore, providing shorter sequences (e.g., siRNA) is more common for mammalian cells. According to one embodiment of the invention, antisense sequences or sequences homologous to microRNAs of naturally occurring RNA molecules targeting the 3'UTR of proteins can also be envisioned as RNAi sequences.

[0169] Therefore, in the case of a DNA system, the polynucleotide used to interfere with RNA is a deoxyribonucleotide, which, when transcribed into RNA precursor ribonucleotides, provides a dsRNA as described above.

[0170] When locating the polynucleotides to minimize interference with alternative splicing, polynucleotides for interfering RNA are particularly preferred. This can be achieved by locating these polynucleotides distal to the alternative splicing control sequence, preferably to the control sequence 3'. In another preferred embodiment, substantially self-complementary regions can be separated from each other by one or more splicing control sequences mediating alternative splicing, such as introns. Preferably, the self-complementary regions are arranged as a series of two or more inverted repeats, each inverted repeat separated by a splicing control sequence (preferably an intron), as defined elsewhere.

[0171] In this configuration, different alternatively spliced ​​transcripts can have essentially self-complementary regions separated by non-self-complementary sequences of varying lengths in the mature (post-alternatively spliced) transcript. It should be understood that essentially self-complementary regions are those capable of forming hairpins, for example, because a portion of the sequence is able to pair bases with other portions of the sequence. These two portions do not need to be perfectly complementary to each other, as there may be some mismatches or tolerances within each portion where segments do not pair bases with each other. Such segments may not have equivalents in other portions, thus losing symmetry and forming a “protruding” form, which is generally known from base pair complementarity.

[0172] In another preferred embodiment, one or more segments of a sequence substantially complementary to another portion of the primary transcript are located relative to at least one splicing control sequence, such that they are not included in all transcripts produced by alternative splicing of the primary transcript. This method produces some transcripts that are predisposed to producing dsRNA, while others are not. dsRNA can be produced in a sex-specific, stage-specific, germline-specific, or tissue-specific manner, or combinations thereof, mediated by alternative splicing, such as sex-specific, stage-specific, germline-specific, or tissue-specific methods.

[0173] Fusion Leader

[0174] In some embodiments, it is desirable that the target functional protein does not have a splice control module protein sequence. In some embodiments, the splice control module is operatively linked to a polynucleotide encoding a polypeptide that is proteolytically cleaved by a polypeptide that stimulates translation (a "fusion leader sequence" for the polynucleotide and a "fusion leader polypeptide" for the encoded polypeptide). An example of such a fusion leader sequence is a polynucleotide encoding ubiquitin. Such a fusion leader sequence can be operatively linked within a frame to the 3' end of the splice control module and operatively linked within a frame to the gene encoding the target protein (i.e., from 5' to 3': splice control module - fusion leader sequence - target gene). In this case, the splice control module / fusion leader polypeptide is cleaved from the target protein by a specific protease in the cell. Any other similar fusion, besides ubiquitin, can be performed instead of ubiquitin, which will have the effect of stimulating cleavage of the N-terminal splice control module. An example of a polynucleotide encoding ubiquitin is provided as SEQ ID NO:30. The ubiquitin fusion leader can be any polynucleotide encoding a functional ubiquitin leader polypeptide from any organism, provided that the ubiquitin leader is reliably cleaved in an arthropod system. One example is Drosophila ubiquitin (e.g., SEQ ID NO:79), which is cleaved from a functional protein that causes lethal, harmful, or infertile effects.

[0175] Promoter and 5'UTR

[0176] Each splicing module, operatively linked to a gene having lethal, harmful, or sterile effects, is operatively linked to a promoter capable of being activated by an activating or transactivating transcription factor encoded by the gene, which is also included in at least one gene expression system. Preferably, any combination of promoter and splicing control modules is contemplated. Preferably, the promoter is specific to a particular protein having transient temporal or limited spatial effects, such as cell-autonomous effects.

[0177] Promoters can be large or complex, but when introduced into non-host insects, they often suffer from misuse or sporadic application. Therefore, in some embodiments, the smallest promoters are preferred. It should be understood that the smallest promoters can be obtained directly from known promoter sources or derived from larger, naturally occurring, or other known promoters. Suitable smallest promoters and how to obtain them will be apparent to those skilled in the art. For example, suitable minimal promoters include minimal promoters derived from Hsp70, P minimal promoters, CMV minimal promoters, Act5C-based minimal promoters, BmA3 promoter fragments, the Drosophila sryα embryo-specific promoter (Horn and Wimmer (2003) Nat. Biotechnol. 21(1): 64-70) or homologs thereof, or promoters from other embryo-specific or embryo-active genes, such as the promoter of the Drosophila gene slow as molasses (slam) embryo-active gene or homologs from other species, and the Adh core promoter (Bieschke, E. et al. (1998) Mol. Gen. Genet., 258: 571-579). It will be apparent to those skilled in the art how to ensure that the selected promoter is active. Preferably, at least one operable promoter present in this invention is active during the early development of the host organism, particularly during the embryonic stage, to ensure that lethal genes are expressed during the early development of the organism.

[0178] In some implementations, the promoter can be activated by environmental conditions, such as the presence or absence of specific factors like tetracycline (or its analogues) in the tet system described herein, thereby allowing the expression of the target gene to be easily manipulated by a technician. In some implementations, a suitable promoter is the hsp70 heat shock promoter, which, for example, allows the user to control expression by altering the ambient temperature to which the host is exposed in the laboratory or field. Another example of temperature control is described in Fryxell and Miller (1995) J. Econ. Entomol. 88:1221-1232.

[0179] Alternatively, the promoter can be specific to a wider range of proteins or to specific proteins with long-term and / or broad systemic effects, such as hormones, positive or negative growth factors, morphin, or other secreted or cell surface signaling molecules. For example, this would allow for a broader pattern of expression, such that the binding of a morphin promoter to a stage-specific alternative splicing mechanism could result in morphin being expressed only at a certain life cycle stage, but still sensed after that stage (i.e., morphin can still function and have an effect). Preferred examples are morphin / signaling molecules Hedgehog, Wingless / WNT, TGFβ / BMP, EGF, and their homologs, which are well-known evolutionarily conserved signaling molecules.

[0180] It is also foreseeable that promoters activated by a range of protein factors, such as transactivators, or promoters with broad systemic effects, such as hormones or morphogens, can be used in combination with alternative splicing mechanisms to achieve tissue- and sex-specific control or sex- and stage-specific control, or other combinations of stage, tissue, germline, and sex-specific control.

[0181] It is also envisioned that more than one promoter and optional enhancer can be used in this system as alternative means of initiating transcription of the same protein, or because the genetic system contains more than one gene expression system (i.e., more than one gene and its associated promoter).

[0182] In some embodiments, at least one of the promoters is a heat shock promoter, such as Hsp70. Examples of sequences containing the Hsp70 promoter (HSP70 minipro) are SEQ ID NO:18 and SEQ ID NO:41. In other embodiments, at least one of the promoters is the sryα embryo-specific promoter from Drosophila melanogaster (Horn and Wimmer (2003) Nat. Biotechnol. 21(1): 64-70) or a homolog thereof, or an embryo-specific or embryo-active promoter from other species, such as the Drosophila gene slow as molasses (slam) or an embryo-active gene from a homolog of another species. In some embodiments, a promoter based on human CMV minipro is used, with or without other elements, such as tetOx7 and turnip yellow mosaic virus (TYMV) 5'UTR (collectively, the "TRE3G promoter"). An example of a promoter based on hCMV minipro is provided as SEQ ID NO:65. An example of a 5' UTR sequence of turnip yellow mosaic virus (TYMV) is SEQ ID NO:64, and an example of a tetOx7 enhancer sequence is SEQ ID NO:66. Together, these constitute an instance of the TRE3G promoter (SEQ ID NO:63).

[0183] Other useful promoters include, but are not limited to, the IE1 promoter (e.g., SEQ ID NO:26) of the baculovirus *Autographica californica* nucleopolyhedrosisvirus (AcNPV); the Hsp83 promoter; the sryα embryo-specific promoter from *Drosophila melanogaster* (Horn and Wimmer (2003) Nat. Biotechnol. 21(1): 64-70) or its homologs; the promoter from the *Drosophila* gene slow as molasses (slam) or homologs from other species; the β-tubulin promoter; the topi promoter; the aly promoter; the protamine promoter; and actin promoters, such as the insect muscle actin promoter Act5c (WO 2014 / 135604); or the Opie2 promoter from the polynuclear polyhedrosis virus of *Orgyia pseudotsugata*.

[0184] Transcription control elements

[0185] Preferably, the polynucleotide expression system is a recombinant dominant lethal genetic system whose lethality is conditional. Suitable conditions include temperature, for example, so that the system is expressed at one temperature and not expressed at another, or expressed to a lesser extent. Lethal genetic systems may act on specific cells or tissues or have an effect on the entire organism. It will be understood that the term lethality as used herein encompasses all such systems and consequences. Similarly, “kill” and similar terms refer to the effective expression of a lethal system and thus impose a harmful or sex-altering phenotype, such as death.

[0186] More preferably, the polynucleotide expression system is a recombinant dominant lethal genetic system whose lethality is conditional and is not expressed under permissible conditions requiring the presence of substances not present in the organism's natural environment, so that the lethality of the lethal system occurs in the organism's natural environment.

[0187] In some implementations, the encoded sequence encodes lethality associated with systems such as the tet system described in WO 01 / 39599 and / or WO2005 / 012534.

[0188] Indeed, preferably, the expression of the lethal gene is under the control of a repressible transactivator protein. It is also preferred that the gene whose expression is regulated by alternative splicing encodes a transactivator protein, such as tTA, or a variant thereof, such as tTAV2 or tTAV3. Non-limiting examples of polynucleotides encoding tTAV proteins and variants include SEQ ID NO:10 (tTAV); SEQ ID NO:81 (tTAV2) and SEQ ID NO:82 (tTAV3). The proteins encoded by them are provided as SEQ ID NO:80 (tTAV), SEQ ID NO:97 (tTAV2), and SEQ ID NO:98 (tTAV3). This does not contradict the fact that the regulatory protein is lethal. In fact, both are particularly preferred. In this respect, we particularly prefer the system to include a positive feedback system as taught in WO2005 / 012534.

[0189] Preferably, the lethal effect of the dominant lethal system is conditionally suppressable. In some embodiments, it exerts its lethal effect only in females. In other embodiments, the lethal effect works only in males; that is, the lethal effect is expressed in either males or females (as needed). For example, if a dominant lethal system is present in insects, it preferably causes the death of at least 40% of the insects. In some embodiments, without inhibitors, it causes the death of at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the insects in the genetic system.

[0190] Therefore, in some embodiments, one or more dominant lethal genes are tTA or tTAV gene variants, and the enhancer is a tetO element containing one or more tetO operator gene units. When binding to the product of a tTA or tTAV gene variant, tetO is able to enhance the transcriptional level of the promoter in its vicinity upstream of the promoter in either direction. In some embodiments, each enhancer is independently one of tetOx1, tetOx2, tetOx3, tetOx4, tetOx5, tetOx6, tetOx7, tetOx8, tetOx9, tetOx10, tetOx11, tetOx12, tetOx13, tetOx14, tetOx15, tetOx16, tetOx17, tetOx18, tetOx19, tetOx20, and tetOx21. In some embodiments, each enhancer is independently one of tetOx1, tetOx7, tetOx14, and tetOx21. In embodiments containing more than one enhancer, the first enhancer may be the same as or different from the second enhancer. Examples of the tetOx7 element are shown in SEQ ID NO:20, SEQ ID NO:42, and SEQ ID NO:66. An example of tetOx14 is shown in SEQ ID NO:83. An example of the tetOx21 element is shown in SEQ ID NO:84.

[0191] Other components

[0192] In some implementations, the system includes additional upstream, 5' factors and / or downstream 3' factors for controlling expression. Examples include enhancers, such as fat-body enhancers from the Drosophila vitellin gene, and homologous region (hr) enhancers from baculoviruses, such as AcNPV Hr5 (SEQ ID NO:27 or SEQ ID NO:49). It should also be understood that the RNA product will include, for example, suitable 5' and 3' UTRs. Examples of 5' and 3' UTRs include, but are not limited to, TYMV 5' UTR (SEQ ID NO:64); Drosophila melanogaster fs(1)K10 3' UTR (SEQ ID NO:19); SV40 3' UTR (SEQ ID NO:43), P10 3' UTR (SEQ ID NO:28 or SEQ ID NO:50); or any other suitable 5' or 3' UTR that functions in the expression system.

[0193] It should be understood that, with reference to the start and stop codons, a polynucleotide sequence to be expressed in an organism is defined between the start and stop codons, but this does not preclude the location of at least one splicing control sequence, its elements, or other sequences, such as introns, in this region. Indeed, it will be apparent from this specification that, in some embodiments, the splicing control sequence may be located in this region.

[0194] Furthermore, in some embodiments, for example, the splicing control sequence may overlap with the start codon at least in the sense that the G in the ATG can be the starting 5' G of the splicing control sequence. Therefore, the term "between" can be considered to refer to the sequence from the start of the start codon (3' to the start nucleotide, i.e., A), preferably 3' to the second nucleotide of the start codon (i.e., T), upwards to the 5' side of the first nucleotide of the stop codon. Alternatively, as will be apparent from a simple reading of the polynucleotide sequence, a stop codon may also be included.

[0195] Combination of other expression units

[0196] The present invention also provides multiple expression units. In some embodiments, a first expression unit includes a dsx splicing module for expressing transcription factors such as tTAV, tTAV2, tTAV3, tTAF, or any analogue thereof. The expression unit includes a recognition sequence for the transcription factor such that, in the absence of tetracycline or tetracycline analogues, expression of the transcription factor leads to positive feedback to drive further expression of transcription factors that are lethal or harmful to arthropods.

[0197] In other embodiments, the first expression unit includes a dsx splicing module for expressing transcription factors that may or may not have harmful or lethal effects, but act on the second expression unit to drive the transcription of a functional protein or nucleic acid that has harmful, lethal, or sterile effects (e.g., Hid or its homologs, Reaper (Rpr) or its homologs, Nipp1Dm or its homologs, calmodulin or its homologs, Michelob-X or its homologs, tTAV or its homologs, tTAV2 or its homologs, tTAV3 or its homologs, tTAF or its homologs, Medea or its homologs, microRNA toxins, or nucleases (e.g., EcoRI, FokI, etc.), and optionally drives the expression of additional transcription factors from the first expression unit (i.e., positive feedback). In this way, arthropods splice dsx / transcription factor expression units in a sex-specific manner. The primary transcript of the first expression unit is expressed by transcription factors that drive the expression of a second expression unit in one sex but not in the other, and optionally drive the expression of other transcription factors via positive feedback. In some embodiments, the first expression unit produces tTAV or its homologs, tTAV2 or its homologs, tTAV3 or its homologs, tTAF or its homologs, and is under the control of tetracycline-responsive transcriptional control elements such as tetO. The second transcription unit produces a protein with harmful, lethal, or sterile effects. In some embodiments, one or both expression units include a splicing module. Preferably, transcription from the first expression unit can be repressed in the presence or absence of a chemical ligand. The second expression unit can also be regulated in a sex-specific manner by adding a second splicing control module, which may be the same as or different from the first splicing control module, as long as it functions in arthropods. Other splicing control modules have been described, for example, in WO 2018 / 029534 and WO 2007 / 091099.

[0198] Marker protein

[0199] The expression system of the present invention may further comprise a polynucleotide encoding a marker protein, which can be expressed to identify arthropods (e.g., insects) containing the expression system. Such a polynucleotide may be operatively linked to 5' and / or 3' elements to aid expression. For example, a promoter and optionally an enhancer may be operatively linked to the polynucleotide encoding the marker protein. This promoter may be the same as or different from a promoter used to express a gene having lethal, harmful, or sterile effects. Examples of promoters that may be used include constitutive promoters that cause constitutive expression of the marker protein. Examples of useful promoters include, but are not limited to, the IE1 promoter (e.g., SEQ ID NO:26) of the baculovirus autographicacalifornica nucleopolyhedrosisvirus (AcNPV); the Hsp83 promoter; the sryα embryo-specific promoter from Drosophila melanogaster (Horn and Wimmer (2003) Nat. Biotechnol. 21(1): 64-70) or homologs thereof; the promoter from the Drosophila gene slow asmolasses (slam) or homologs from other species; the β-tubulin promoter; the topi promoter; the aly promoter; the protamine promoter; and the actin promoter. In some embodiments, the promoter is the IE1 promoter (e.g., SEQ ID NO:26). The expression system marker polynucleotide / promoter may further include an enhancer. Suitable enhancers may include, but are not limited to, the baculovirus Autographicacalifornica nucleopolyhedrosisvirus (AcNPV) Hr5 enhancer (e.g., SEQ ID NO:27 or SEQ ID NO:49), tetOx1, tetOx2, tetOx3, tetOx4, tetOx5, tetOx6, tetOx7, tetOx8, tetOx9, tetOx10, tetOx11, tetOx12, tetOx13, tetOx14, tetOx15, tetOx16, tetOx17, tetOx18, tetOx19, tetOx20, and tetOx21. In some embodiments, each enhancer is independently one of tetOx1, tetOx7, tetOx14, and tetOx21. In embodiments that include more than one enhancer, the first enhancer may be the same as or different from the second enhancer. Examples of the tetOx7 element are shown in SEQ ID NO:20, SEQ ID NO:42 and SEQ ID NO:66.An example of tetOX14 is shown in SEQ ID NO:83. An example of the tetOx21 element is shown in SEQ ID NO:84.

[0200] The labeled protein can be a protein that confers drug resistance or a fluorescent protein. Examples of fluorescent proteins that can be used as labeled proteins include, but are not limited to, AmCyan, Clavularia, ZsGreen, ZsYellow, Discosomastriata, DsRed2, AsRed, Discosoma Green, Discosoma Magenta, HcRed-2A, mCherry, green fluorescent protein (GFP), red fluorescent protein (RFP), and HcRed-Cr1-tandem, or one or more mutants or variants thereof. As shown in the example below, DsRed2 (Clontech) can be used. Examples of polynucleotide sequences encoding DsRed2 are provided as SEQ ID NO:1, SEQ ID NO:23, and SEQ ID NO:45. The polypeptide sequence encoded by SEQ ID NO:1 (DsRed2) is provided as SEQ ID NO:85.

[0201] Introducing constructs into organisms

[0202] Methods for introducing or transforming gene system constructs and inducing expression in relevant organisms are well known in the art. It should be understood that the system or construct is preferably administered as a plasmid, but is typically tested after integration into the genome. Plasmid vectors can be introduced into desired host cells by methods known in the art, such as transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipid transfection (lysosomal fusion), using a gene gun or DNA vector transporter (see, for example, Wu et al., (1992) J. Biol. Chem. 267:963; Wu et al. (1988) J. Biol. Chem. 263:14621; and Canadian patent application No. 2,012,311 of Hartmut et al.). Intraembryonic administration via microinjection is a preferred method for producing genetically engineered arthropods (e.g., insects). Plasmids can be linearized before or during administration. Plasmid vectors can be integrated into the host chromosome by any known method. Well-known locus-specific insertion methods can be used, including homologous recombination and recombinase-mediated genome insertion. In another embodiment, locus-specific insertion can be performed via recombinase site-specific gene insertion. In one example, the piggyBac sequence can be incorporated into a vector to drive vector insertion into the host cell chromosome. Other techniques, such as CRISPR, TALEN, and AttP / AttB recombination, can also be used. Not all plasmids can be integrated into the genome. In cases where only a portion of the plasmid is integrated into the genome, preferably, this portion includes at least one splicing control module capable of mediating alternative splicing.

[0203] Genetically engineered insects

[0204] The vectors of the present invention can be used to produce transgenic insects of the genera *Spodoptera*, *Helicoverpa*, *Chrysodeixis*, *Anticarsia*, *Peridroma*, and *Heliothis*. Examples of genetically engineered insect species that may result include, but are not limited to, the fall armyworm (Spodoptera frugiperda), the beet armyworm (Spodoptera exigua), the African cotton leafworm (Spodoptera littoralis), the cotton bollworm (Helicoverpa armigera), the spotted leafworm (Peridroma saucia), the soybean leafworm (Helicoverpa zea; other common names include cotton bollworm and tomato leafworm), the soybean leafworm (Chrysodeixis includens), the velvet bean caterpillar (Anticarsia gemmatalis), and the tobacco leafworm (Heliothis virescens).

[0205] Specific implementation plans (pOX5403, pOX5368, and pOX5382)

[0206] In certain embodiments, the present invention provides a splicing cassette containing exons and introns derived from the bisexual (dsx) gene of the fall armyworm. The splicing cassette includes exons 2, 3, 3a, 4, 4b, and 5 of dsx in various arrangements, as well as introns 2, 3, and 4. In some embodiments, male splicing occurs from exons 2 to 5. Therefore, for male-specific splicing, exons 2 and 5 must be included. Female splicing can be performed by linking exons 2 and 3 with heterologous sequences encoding lethal, harmful, or sterile functional proteins. For these embodiments, exons 2 and 3, as well as intron 2 (see...), are required. Figure 6 Therefore, differential splicing can be performed using exons 2, 3, and 5 with introns 2 and 4. Female splicing can also be accomplished by joining exons 2, 3, 4, and 5 or exons 2, 3, 3a, 4, and 5 (see [link to original text]). Figure 3 and Figure 9 Therefore, in these constructs, differential splicing can be performed using exons 2, 3, 4 and 5 or exons 2, 3, 3a, 4 and 5 (and optional exon 4b) and introns 2, 3 and 4.

[0207] These implementations allow the construction to link a splice cassette to a heterologous gene of interest, such as a gene conferring lethality, like the tTAV gene, and optionally to a 5' leader sequence, such as ubiquitin (see [link to documentation]). Figure 3 and Figure 9 Alternatively, the heterologous sequence can be placed between the elements of the splice cassette, such that the female splices the primary transcript of the splice cassette to include the heterologous sequence within the frame, while the male splices both the primary transcript and the heterologous sequence of the splice cassette to splice the heterologous sequence (see [link to original text]). Figure 6 ).

[0208] For these constructs, the exons encode the following amino acid sequences: exon 2 (SEQ ID NO:71), exon 3 (SEQ ID NO:72), exon 3a (SEQ ID NO:73), exon 4 (SEQ ID NO:74), and exon 5 (SEQ ID NO:75). In specific embodiments, the polynucleotide sequences of the exons and introns are as follows: exon 2 (SEQ ID NO:7 or SEQ ID NO:32); and exon 3 (SEQ ID NO:94, SEQ ID NO:34 or SEQ ID NO:56); exon 3a (SEQ ID NO:12); exon 4 (SEQ ID NO:15), exon 4b (SEQ ID NO:14) (the exon 4b / exon 4 sequences are shown in SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92), exon 5 (SEQ ID NO:17), intron 2 (SEQ ID NO:55), intron 3 (SEQ ID NO:58) and intron 4 (SEQ ID NO:39). The ubiquitin leader sequence in these constructs has the polynucleotide sequence of SEQ ID NO:30 or SEQ ID NO:52.

[0209] These specific implementations have operative connections to the tetO enhancer sequence (in Figure 2 , Figure 5 and Figure 8 The tetO enhancer sequence is the tetOx7 enhancer, which is the Hsp70 minipro promoter of Drosophila melanogaster or the human CMV minipro (with a TYMV 5'UTR) (shown as pOX5403, pOX5368, and pOX5382, respectively). The SEQ ID NO of the polynucleotide sequences of these elements is shown in Tables 1, 2, and 3.

[0210] Methods to suppress arthropod / insect populations and reduce crop damage

[0211] The present invention also provides a method for suppressing wild arthropod populations (e.g., noctuid moths) by releasing genetically engineered male arthropods (e.g., noctuid moths) comprising the expression system of the present invention into wild arthropod populations of the same species. The genetically engineered arthropods are then mated with wild arthropods, and the offspring of this mating possess primary transcripts of differentially spliced ​​splice cassettes to produce functional proteins (for female arthropods) with lethal, harmful, or sterile effects, leading to the death of female offspring or their inability to reproduce effectively, thereby suppressing the wild arthropod population.

[0212] Insects can be reared for reproduction by containing compounds that inhibit the expression of functional proteins and rescue them from lethal, harmful, or sterile effects, thus producing more adult insects. When only male insects are reared for release, the compounds that inhibit functional proteins are eliminated, and the female insects will die or be unable to reproduce because they will produce the functional proteins. Even in the absence of the inhibitory compounds, male insects that do not produce functional proteins will survive without any adverse effects.

[0213] The present invention also provides a method for reducing, suppressing, or eliminating crop damage caused by arthropods (e.g., noctuid moths), comprising releasing genetically engineered male arthropods (e.g., noctuid moths) containing the expression system of the present invention into a wild arthropod population of the same species, and then mating the genetically engineered arthropods with the wild arthropods. The offspring of this mating differentially splice the primary transcripts of the splice cassette, thereby producing functional proteins (for female arthropods) with lethal, harmful, or sterile effects, causing the death of female offspring or the inability of female offspring to reproduce effectively, thereby suppressing the wild arthropod population and reducing, suppressing, or eliminating crop damage caused by wild insects.

[0214] This invention also provides a method for resistance management in Noctuidae insects, comprising releasing genetically engineered male Noctuidae insects containing the expression system of this invention into a wild population of the same species, wherein the population contains a variety of insects resistant to pesticides and biological pesticides (e.g., Bt-type), and then mating the genetically engineered insects with wild insects. The offspring of this mating have primary transcripts of differentially spliced ​​splice cassettes to produce functional proteins (for female Noctuidae insects) that have lethal, harmful, or sterile effects, resulting in the death of female offspring or their inability to reproduce effectively. Male offspring surviving from such mating with wild females also effectively transmit susceptible alleles present in the transgenic population (i.e., the trait is infiltrated into the wild population) and dilute the resistance frequency of the wild pest population. Further description of this strategy can be found, for example, in WO2004098278. In this way, the method suppresses wild Noctuidae insect populations and mitigates or reverses pesticide resistance in wild Noctuidae insect populations.

[0215] The present invention also includes a method for detecting genetically engineered insects comprising the female gene expression system of the present invention, which is achieved by expressing a reporter gene (e.g., but not limited to, a fluorescent protein) by including a reporter gene expression unit in the expression system, wherein the expression of the reporter gene in the system is detectable.

[0216] In some embodiments, the reporter gene is a fluorescent protein. In some embodiments, the fluorescent protein is DsRed2 (e.g., encoded by SEQ ID NO:1 and having the amino acid sequence of SEQ ID NO:80). In some embodiments, the reporter gene is detected by examining the insect under light of a specific wavelength.

[0217] Example

[0218] The following examples relate to constructs based on the dsx gene of the fall armyworm (Noctuidae). To allow open reading frames, reduce the likelihood of internal translation start sites, control the size of expressed fragments, and generate reliable sex-specific splicing between males and females, some exons and introns were modified.

[0219] The dsx used in the splice cassette and expression system of this invention completely eliminates exon 1. Exon 2 is truncated by approximately 75% and a 5-nucleotide addition (atgaa) is added to the 5' end to provide the initiating methionine and retain the exon within the reading frames of OX5403 and OX5382. The entire exon 3 and exon 3a are retained in OX5382 and OX5403, with another g added to the 3' end to retain the reading frame. In OX5368, the tTAV protein-coding sequence, containing the start and stop codons, is placed within exon 3 linked by a polynucleotide linker (see [link to dsx]). Figure 19 Although the entire exon 4b and exon 4 are preserved, only exon 4 is spliced ​​into the functional protein due to a splicing event that occurs within the coding region of exon 4b / 4. The entire exon 5 is used together with an additional 6 nucleotides (gtagcg) provided at the 3' end of exon 5.

[0220] In addition, the following point mutations were introduced for pOX5382 and pOX5403 (numbered with reference to endogenous dsx cDNA, starting from the beginning of exon 1):

[0221]

[0222] Similarly, in addition to the above truncation, the following point mutations were designed for pOX5368 (numbered with reference to endogenous dsxcDNA, starting from the beginning of exon 1):

[0223]

[0224] Example 1. Production of the fall armyworm OX5403

[0225] plasmid pOX5403 ( Figure 1 This plasmid is based on the cloning vector pKC26-FB2 (Genbank #HQ998855). The plasmid backbone contains the pUC origin of replication and the β-lactamase gene conferring ampicillin resistance, used in molecular cloning procedures. This plasmid portion is not included in the rDNA or integrated into the insect genome.

[0226] pOX5403 also contains complete rDNA incorporated from insects, including a synthetic DNA sequence (Clontech) encoding the DsRed2 red fluorescent marker protein, a synthetic DNA sequence of the tetracycline-repressible transcriptional activator tTAV (based on a sequence fusion from E. coli and the HSV-1VP16 transcriptional activator), and a modified Sfdsx splicing module derived from the fall armyworm. Figure 2 The components shown are listed in detail in Table 1. Plasmids are prepared using standard DNA cloning procedures.

[0227] The first gene is the DsRed2 gene, controlled by the Hr5 / IE1 promoter. This gene is responsible for producing the DsRed2 fluorescent protein, which serves as a visual marker for integrating rDNA into the fall armyworm genome and identifying transgenic insects.

[0228] The second gene is the Sfdsx_tTAV gene, controlled by the complex promoter (TRE3G), which includes a truncated version of the hCMV minimal promoter fused to the TYMV 5'UTR, located downstream of the tetracycline-responsive operator (tetOx7) (Loew et al. (2010) BMC Biotechnol. 10:81). The Sfdsx splicing module enables female-specific expression of the tTAV protein.

[0229] The Sfdsx_tTAV gene is expressed in a female-specific manner by including a portion of the fall armyworm bisexual gene (Sfdsx). This gene is transcribed into three different sex-specific alternatively spliced ​​transcripts: two female-specific (F1 and F2) and one male-specific (M) transcript. Figure 3 The variations in these three transcripts are due to sex-specific differences in the mRNA sequences, resulting from sex-specific splicing of the RNA encoded by the Smdsx sex-specific alternative splicing module. In the F1 and F2 transcripts, the sequence encoding tTAV is within the reading frame of the upstream start codon. Figure 2 In female transcripts, splicing occurs to frame-link exons 2, 3, 4, and 5 with the ubiquitin leader and tTAV sequence, thereby translating the tTAV sequence and cleaving it from the translated protein, or frame-linking exons 2, 3 / 3a, 4, and 5 with the ubiquitin leader and tTAV sequence, causing the tTAV sequence to be translated and cleaved from the translated protein. In M transcripts, the exclusion of dsx exons 3, 3a, 4b, and 4 prevents the production of the tTAV protein because the tTAV coding sequence is not within the frame containing the tTAV start codon, but is within the frame with a stop codon downstream of exon 5, which precedes the tTAV coding sequence. Therefore, M transcripts contain a stop codon within the frame in their coding sequence, which may lead to M transcript mRNA degradation through nonsense-mediated decay (Hansen et al. (2009) PLoS Genet. 5:e1000525).

[0230] Plasmid pOX5403 contains complete rDNA incorporated from insects, including a synthetic DNA sequence encoding the DsRed2 red fluorescent marker protein, a synthetic DNA sequence of the tetracycline-inhibitory transcription activator tTAV (based on a sequence fusion of E. coli and the HSV-1VP16 transcription activator), and a modified Sfdsx splicing module derived from the fall armyworm.

[0231] Table 1. Genetic components of OX5403

[0232]

[0233]

[0234]

[0235] Transformation was performed using a non-autonomous piggyBac transposon element, first described in (Thibault et al., (1999) Insect Mol. Biol. 8:119-123), co-injected with a non-integrative source of the piggyBac transposase (mRNA transcribed in vitro from plasmid pOX3022). This piggyBac transposon was originally isolated from cell cultures of Trichoplusia ni and has been used in transformations of several insects (Diptera, Lepidoptera, Coleoptera) (Handler, (2002) Proc. Natl. Acad. Sci. USA 95:7520-7525; O'Brochta et al., (2003) J. Exp. Biol. 206:3823-3834; Tamura et al., (2000) Nat. Biotechnol. 18:81-84). As originally described, the transposon consists of two components: a coding sequence for the piggyBac transposase and a terminal inverted repeat sequence that is recognized and processed by the transposase for integration into the target DNA. However, the minimum piggyBac element used to integrate OX5382 rDNA into the genome of the grass armyworm is based on the minimum sequence (including but not limited to the terminal inverted repeat sequence) required for efficient integration of the piggyBac transposase into the target DNA, and does not contain the coding sequence required to generate the piggyBac transposase (Li et al. (2005) Insect Mol. Biol. 14:17-30).

[0236] Example 2. Production of the fall armyworm OX5368

[0237] plasmid pOX5368 ( Figure 4 This plasmid is based on the cloning vector pKC26-FB2 (Genbank #HQ998855). The plasmid backbone contains the pUC origin of replication and the β-lactamase gene, which confers ampicillin resistance, and is used in molecular cloning procedures. This plasmid portion is not included in the rDNA or integrated into the insect genome.

[0238] Plasmid pOX5368 also contains complete insect rDNA, including a synthetic DNA sequence encoding the DsRed2 red fluorescent marker protein, a synthetic DNA sequence of the tetracycline-inhibited transcriptional activator tTAV2 (based on a fusion of the sequences of E. coli and the HSV-1VP16 transcriptional activator), and a modified Sfdsx splicing module derived from the fall armyworm. Figure 5The components shown are listed in detail in Table 2. Plasmids were prepared using standard DNA cloning methods.

[0239] The Sfdsx_tTAV2 gene was expressed in a female-specific manner by incorporating a portion of the fall armyworm's bisexual gene (Sfdsx). This gene was transcribed into three distinct sex-specific alternative splicing transcripts: two female-specific (F1 and F2) transcripts and one male-specific (M) transcript. Figure 6 The variations in these three transcripts are due to the sex-specific inclusion of different mRNA sequences, which are caused by sex-specific splicing of the RNA encoded by the Sfdsx sex-specific alternative splicing module. In the F1 and F2 transcripts, the mRNA sequence encoding tTAV2 is spliced ​​with exon 2, exon 3 5' portions within the frame and translated into the tTAV2 protein. Figure 6 In the M transcript, excluding exons 3, 3a, 4b, and 4 of the dsx splice transcript prevents the production of the tTAV2 protein because the tTAV2 coding sequence is spliced ​​entirely from the mRNA.

[0240] Table 2. Genetic components of OX5368

[0241]

[0242]

[0243] Transformation using a non-autonomous piggyBac transposon element was first described in (Thibault et al., 1999) by co-injection with a non-integrative piggyBac transposase (mRNA transcribed in vitro from plasmid pOX3022). This piggyBac transposon was originally isolated from cell cultures of the white armyworm (Trichoplusia ni) and has been used for transformation in various insects (Diptera, Lepidoptera, Coleoptera) (Handler, 2002; O'Brochta et al., 2003; Tamura et al., 2000). As originally described, the transposon consists of two components: a coding sequence encoding the piggyBac transposase and a terminal inverted repeat sequence recognized and processed by the transposase for integration into the target DNA. However, the minimum piggyBac element used to integrate OX5368 rDNA into the genome of the grass armyworm is the minimum sequence (including but not limited to terminal inverted repeat sequences) required for efficient integration of the piggyBac transposase into the target DNA, and does not contain the coding sequence required to generate the piggyBac transposase (Li et al., 2005).

[0244] Example 3. Production of the fall armyworm OX5382

[0245] plasmid pOX5382 ( Figure 7 Based on the cloning vector pKC26-FB2 (Genbank#HQ998855). The plasmid backbone contains the pUC origin of replication and the β-lactamase gene conferring ampicillin resistance, and can be used in molecular cloning procedures. This plasmid portion is not included in rDNA or integrated into the insect genome.

[0246] pOX5382 also contains complete rDNA incorporated from insects, including a synthetic DNA sequence encoding the DsRed2 red fluorescent marker protein, a synthetic DNA sequence of the tetracycline-repressible transcription activator tTAV (based on a fusion of sequences from E. coli and the HSV-1VP16 transcription activator), and a modified Sfdsx splicing module derived from the fall armyworm. Figure 8 The components shown are listed in detail in Table 3. The plasmid was prepared by Oxitec Ltd using standard DNA cloning procedures.

[0247] The Sfdsx_tTAV gene is expressed in a female-specific manner by containing a portion of the fall armyworm's bisexual gene (Sfdsx). This gene is transcribed into three distinct sex-specific alternative splicing transcripts: two female-specific (F1 and F2) transcripts and one male-specific (M) transcript. Figure 9 The variations in these three transcripts are due to the sex-specific inclusion of different mRNA sequences, which are the result of sex-specific splicing of RNA encoded by the Sfdsx sex-specific alternative splicing module. In the F1 and F2 transcripts, the sequence encoding tTAV is within the reading frame of the upstream start codon ( Figure 9 In the M transcript, the exclusion of dsx exons 3, 3a, 4b, and 4 prevents the production of the tTAV protein because the tTAV coding sequence is not in the same frame as the tTAV start codon and is in the same frame as the stop codon located downstream of exon 5 preceding the tTAV coding sequence. Therefore, the M transcript contains an in-frame stop codon in its coding sequence, which may lead to the degradation of the M transcript mRNA due to nonsense-mediated decay (Hansen et al., 2009).

[0248] Table 3. Genetic components of OX5382

[0249]

[0250]

[0251]

[0252] Transformation using a non-autonomous piggyBac transposon element was first described in (Thibault et al., 1999) and was injected together with a non-integrative piggyBac transposase (mRNA transcribed in vitro from plasmid pOX3022). This piggyBac transposon was originally isolated from cell cultures of the white-spotted armyworm and has been used in transformations in various insects (Diptera, Lepidoptera, Coleoptera) (Handler, 2002; O'Brochta et al., 2003; Tamura et al., 2000). As originally described, the transposon consists of two components: a coding sequence encoding the piggyBac transposase and a terminal inverted repeat sequence recognized and processed by the transposase for integration into the target DNA. However, the minimum piggyBac element used to integrate OX5368 rDNA into the genome of the grass armyworm is the minimum sequence (including but not limited to terminal inverted repeat sequences) required for efficient integration of the piggyBac transposase into the target DNA, and does not contain the coding sequence required to generate the piggyBac transposase (Li et al., 2005).

[0253] The female and male transcripts of the wild-type fall armyworm are in Figure 16 Figure C schematically illustrates that the endogenous stop codon prevents the translation of the spliced ​​primary transcript, except in the case of the male fall armyworm. The splicing of exons in the wild-type fall armyworm and related noctuid moths (cotton bollworms) is respectively... Figure 16 As shown in B and 16A, these related noctuid families splice exons in a highly conserved manner.

[0254] Figure 17 The amino acid sequences of exons 2, 3, 3a, 4, and 5 encoded by female (F) and male (M) transcripts of dsx in constructs OX5403, OX5368, OX5382, endogenous wild-type fall armyworm (endogenous), and cotton bollworm (HA) are shown. Because cotton bollworm and wild-type fall armyworm have a stop codon in exon 3, females do not translate exons 3a, 4b, 4, or 5. The constructs of this invention introduce variations to open the reading frames of exons 3, 3a, 4, and 5, allowing females to translate the entire exon set, although the translation of exon 5 is in a different reading frame than the male transcript and results in a different amino acid sequence.

[0255] Example 4. Penetration of traits in genetically modified fall armyworm

[0256] To assess the penetrance and repression of early sexually exclusive self-limiting traits in OX5403, OX5368, and OX5382, testcrosses were performed between hemizygous males of OX5403, OX5368, and OX5382 and wild-type female moths. First-instar larvae were collected from these hybrids and reared in single cells, fed diets containing 100 μg / ml doxycycline (“with doxycycline”) or without doxycycline (0 μg / ml) (“without doxycycline”). Four classes of moths were expected from these hybrids: (1) male self-limiting moths; (2) female self-limiting moths; (3) wild-type male moths; and (4) wild-type female moths. All classes would survive on doxycycline if the self-limiting trait had good penetrance, but female self-limiting moths would die in the absence of doxycycline. Figure 10 Several substrains of OX5403, OX5368, and OX5382 were tested. Strains meeting the penetrance criteria were selected for further development. Results for each strain (OX5368C, OX5403A, OX5382G, and OX5382J) are shown below. Figure 11 , Figure 12 , Figure 13 and Figure 14 .

[0257] Figure 11 The study showed that OX5368C females carrying the self-limiting trait were fully viable in the presence of doxycycline, but none of them survived to adulthood in the absence of doxycycline. Similarly, Figure 12 It was shown that OX5403A females carrying the self-limiting trait were fully viable in the presence of doxycycline, but no OX5403A females survived to adulthood in the absence of doxycycline.

[0258] To achieve penetrance, two strains of OX5382, OX5382G and OX5382J, were selected. Similar to OX5403A and OX5368C, females of OX5382G and OX5382J carrying the self-limiting trait could survive in the presence of doxycycline (although slightly less than wild-type females), but in the absence of doxycycline, no females of OX5382G or OX5382J survived to adulthood (they were respectively...). Figure 13 and Figure 14 ).

[0259] Example 5. Evaluation of fluorescence during the life cycle of the noctuid moth

[0260] The transgenic strains carrying the self-limiting gene construct also carry and express the fluorescent protein DsRed2 (Clontech; Matz, MV et al. (1999) Nature Biotechnol. 17: 969-973; Lukyanov et al. (2000) J. Biol. Chem. 275(34): 25879).

[0261] The expression of the DsRed2 transgene in the fall armyworm was assessed by examining the DsRed2 fluorescence of early-instar larvae, late-instar larvae, pupae, and adults of both transgenic and wild-type fall armyworms using a Leica M80 microscope equipped with a detection filter: maximum excitation 563 nm, emission 582 nm. Results are shown in... Figure 15 DsRed2 fluorescence was detected in all life stages of the fall armyworm.

[0262] Sequence List Free Text

[0263] SEQ ID NO:1: A variant of a red fluorescent protein from coral (Discosoma) (clontech)

[0264] SEQ ID NO:2: Synthetic DNA

[0265] SEQ ID NO:6: Synthetic DNA based on the sequence of fall armyworm

[0266] SEQ ID NO:10: Optimized fusion tetracycline transactivator protein

[0267] SEQ ID NO:20: Synthetic DNA containing 7 repeat sequences of the Tn10 tet-operon

[0268] SEQ ID NO:22: Synthetic DNA

[0269] SEQ ID NO:23: A variant of a red fluorescent protein from coral (Discosoma) (Clontech)

[0270] SEQ ID NO:24: Synthetic DNA

[0271] SEQ ID NO:29: Optimized fusion tetracycline transactivator protein

[0272] SEQ ID NO:31: Synthetic DNA based on the fall armyworm

[0273] SEQ ID NO:42: Synthetic DNA containing 7 repeat sequences of the Tn10 tet-operon

[0274] SEQ ID NO:44: Synthetic DNA

[0275] SEQ ID NO:45: A red fluorescent protein variant from coral (Discosoma) (Clontech)

[0276] SEQ ID NO:46: Synthetic DNA

[0277] SEQ ID NO:51: Optimized fusion tetracycline transactivator protein

[0278] SEQ ID NO:53: Synthetic DNA based on the sequence of the fall armyworm.

[0279] SEQ ID NO:63: Seven repeat sequences based on the TYMV, hCMV and Tn10 tet operons

[0280] SEQ ID NO:64: Synthetic non-coding fragment based on TYMV sequence

[0281] SEQ ID NO:66: The synthesized DNA contains 7 repeat sequences of the Tn10 tet-operon.

[0282] SEQ ID NO:67: tTAV2

[0283] SEQ ID NO:68: tTAV3

[0284] SEQ ID NO:80: tTAV

[0285] SEQ ID NO:81: tTAV2

[0286] SEQ ID NO 82: tTAV3

[0287] SEQ ID NO:83: The synthesized DNA contains 14 repeat sequences of the Tn10 tet-operon. SEQ ID NO:84: The synthesized DNA contains 21 repeat sequences of the Tn10 tet-operon.

[0288] SEQ ID NO:85: Red fluorescent protein variant from coral (Discosoma) (Clontech) SEQ ID NO:86: Plasmid construct for expression in arthropods

[0289] SEQ ID NO:87: Plasmid construct for expression in arthropods

[0290] SEQ ID NO:88: Plasmid construct for expression in arthropods

[0291] SEQ ID NO:95: Synthetic DNA adapter

[0292] SEQ ID NO:96: Synthetic DNA adapter

[0293] SEQ ID NO:97: tTAV2

[0294] SEQ ID NO:98: tTAV3

[0295] SEQ ID NO:99:tTAV2 ORF

[0296] SEQ ID NO:100: tTAV2

[0297] SEQ ID NO:101:tTAV

[0298] SEQ ID NO:102: tTAV

[0299] SEQ ID NO:103: Translation of transcripts from 5403 and 5382

[0300] SEQ ID NO:104: Translation of exon 2 from Endo

[0301] SEQ ID NO:105: Translation of exon 2 from HA

[0302] SEQ ID NO:106: Translation of exon 3F transcript from 5403

[0303] SEQ ID NO:107: Translation of exon 3F transcript from 5382

[0304] SEQ ID NO:108: Translation of exon 3F transcript from Endo 1

[0305] SEQ ID NO:109: Translation of exon 3F transcript from Endo 2

[0306] SEQ ID NO:110: Translation of exon 3F transcript from HA

[0307] SEQ ID NO:111: Translation of exon 4F transcripts from exons 5403 and 5382

[0308] SEQ ID NO:112: Translation of exon 5M transcript from HA

Claims

1. A method for producing an arthropod containing a female-specific gene expression system, comprising introducing the female-specific gene expression system into the arthropod, wherein the female-specific gene expression system comprises: a. Promoter; b. Polynucleotides encoding functional proteins, whose coding sequence is confined between the start codon and the stop codon; and c. A splicing control polynucleotide that works synergistically with the spliceosome in the arthropod to sex-specifically mediate the splicing of primary transcripts in the arthropod, wherein the primary transcripts comprise exon 2 or a portion thereof of a noctuid sex gene; exon 3 or a portion thereof of a noctuid sex gene; exon 4 or a portion thereof of a noctuid sex gene; exon 5 or a portion thereof of a noctuid sex gene; intron 2 or a portion thereof of a noctuid sex gene; and intron 4 or a portion thereof of a noctuid sex gene; wherein: (a) The first splicing of a polynucleotide RNA transcript produces a first spliced ​​mRNA product that does not have a continuous open reading frame extending from the start codon to the stop codon; and (b) Alternative splicing of the RNA transcript produces an alternatively spliced ​​mRNA product comprising a continuous open reading frame extending from the start codon to the stop codon.

2. The method according to claim 1, wherein, The polynucleotide encoding the functional protein encodes Hid or a homolog thereof, Reaper (Rpr) or a homolog thereof, Nipp1Dm or a homolog thereof, calmodulin or a homolog thereof, Michelob-X or a homolog thereof, tTAV or a homolog thereof, tTAV2 or a homolog thereof, tTAV3 or a homolog thereof, tTAF or a homolog thereof, medea or a homolog thereof, microRNA toxin or nuclease.

3. The method of claim 2, wherein the polynucleotide encoding the functional protein encodes tTAV or a homolog thereof, tTAV2 or a homolog thereof, tTAV3 or a homolog thereof, or tTAF or a homolog thereof.

4. The method according to claim 3, wherein the functional protein comprises the amino acid sequence of SEQ ID NO:80, SEQ ID NO:97 or SEQ ID NO:

98.

5. The method according to claim 2, wherein the nuclease is FokI or EcoRI.

6. The method of claim 1, wherein the expression system further comprises a 3'UTR or a portion thereof operatively linked to the polynucleotide encoding the functional protein.

7. The method of claim 1, wherein the expression system further comprises a ubiquitin leader sequence at the 5' of the polynucleotide encoding a functional protein.

8. The method according to claim 1, wherein, The polynucleotide encoding the functional protein is located at 3' of exon 2 and within exon 3, such that the flanking portion of the polynucleotide encoding the functional protein is the first part of exon 3 encoding the polynucleotide 5' of the functional protein and the second part of exon 3 encoding the polynucleotide 3' of the functional protein.

9. The method according to claim 1, wherein, The polynucleotide encoding the functional protein is located at the 3' of exon 2 and at least a portion of exon 3.

10. The method according to claim 1, wherein, The polynucleotide encoding the functional protein is located at the 3' of exon 2, exon 3, and exon 5.

11. The method of claim 1, wherein in males the primary transcript is spliced ​​such that translation terminates at the 5' of the polynucleotide encoding the functional protein.

12. The method of claim 1, wherein in males the primary transcript is spliced ​​such that the polynucleotide encoding the functional protein is cut from the primary transcript.

13. The method of claim 1, wherein exon 3 comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:

72.

14. The method of claim 8, wherein the first portion comprises the polynucleotide sequence of SEQ ID NO:94, and the second portion comprises the polynucleotide sequence of SEQ ID NO:

9.

15. The method according to claim 1, wherein, Exon 3 contains a polynucleotide sequence of SEQ ID NO:94, SEQ ID NO:34, or SEQ ID NO:

56.

16. The method according to claim 1, wherein, Exon 2 contains a polynucleotide encoding the amino acid sequence of SEQ ID NO:

71.

17. The method according to claim 1, wherein, Exon 2 contains a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:

32.

18. The method of claim 1, wherein the primary transcript further comprises exon 3a or a portion thereof of a Noctuidae hermaphroditic gene, said exon 3a comprising a polynucleotide encoding the amino acid sequence of SEQ ID NO:

73.

19. The method of claim 1, wherein the primary transcript further comprises exon 3a or a portion thereof of a Noctuidae bisexual gene, said exon 3a comprising the polynucleotide sequence of SEQ ID NO:

12.

20. The method of claim 1, wherein exon 4 comprises a polynucleotide encoding an amino acid sequence of SEQ ID NO:

74.

21. The method of claim 1, wherein exon 4 comprises the polynucleotide sequence of SEQ ID NO:

15.

22. The method of claim 1, wherein the primary transcript further comprises exon 4b or a portion thereof of the Noctuidae bisexual gene, thereby forming exon 4b-exon 4 of the Noctuidae bisexual gene, said exon 4b-exon 4 comprising a polynucleotide sequence of SEQ ID NO:90, SEQ ID NO:91 or SEQ ID NO:

92.

23. The method of claim 1, wherein the primary transcript further comprises exon 4b or a portion thereof of a Noctuidae bisexual gene, said exon 4b comprising the polynucleotide sequence of SEQ ID NO:

14.

24. The method of claim 1, wherein exon 5 comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO:

75.

25. The method of claim 1, wherein exon 5 comprises the polynucleotide sequence of SEQ ID NO:

17.

26. The method of claim 1, wherein the intron 2 comprises the polynucleotide sequence of SEQ ID NO:

55.

27. The method of claim 1, wherein the primary transcript further comprises intron 3 or a portion thereof of a hermaphroditic gene from the Noctuidae family, said intron 3 comprising the polynucleotide sequence of SEQ ID NO:

58.

28. The method of claim 1, wherein the intron 4 comprises the polynucleotide sequence of SEQ ID NO:

39.

29. The method of claim 1, wherein exon 2 comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:71; exon 3 comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:72; exon 4 comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:74; and exon 5 comprises a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO:

75. The primary transcript further comprises at least one exon 3a or a portion thereof of a Noctuidae hermaphroditic gene, wherein the exon 3a comprises a polynucleotide encoding an amino acid sequence of SEQ ID NO:

73.

30. The method of claim 1, wherein exon 2 comprises a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32; the first portion of exon 3 comprises a polynucleotide sequence of SEQ ID NO:94, and the second portion comprises a polynucleotide sequence of SEQ ID NO:9; exon 4 comprises a polynucleotide sequence of SEQ ID NO:15; and exon 5 comprises a polynucleotide sequence of SEQ ID NO:

17. The primary transcript further comprises at least one exon 3a or a portion thereof of a Noctuidae hermaphroditic gene, and the exon 3a comprises the polynucleotide sequence of SEQ ID NO:12, and The primary transcript further comprises at least one exon 4b or a portion thereof of a Noctuidae bisexual gene, and the exon 4b comprises the polynucleotide sequence of SEQ ID NO:

14.

31. The method of claim 1, wherein exon 2 comprises a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32; exon 3 comprises a polynucleotide sequence of SEQ ID NO:34 or SEQ ID NO:56; exon 4 comprises a polynucleotide sequence of SEQ ID NO:15; and exon 5 comprises a polynucleotide sequence of SEQ ID NO:

17. The primary transcript further comprises at least one exon 3a or a portion thereof of a Noctuidae hermaphroditic gene, and the exon 3a comprises the polynucleotide sequence of SEQ ID NO:12, and The primary transcript further comprises at least one exon 4b or a portion thereof of a Noctuidae bisexual gene, and the exon 4b comprises the polynucleotide sequence of SEQ ID NO:

14.

32. The method of claim 1, wherein exon 2 comprises a polynucleotide sequence of SEQ ID NO:7 or SEQ ID NO:32; exon 3 comprises a polynucleotide sequence of SEQ ID NO:34 or SEQ ID NO:56; exon 4 comprises a polynucleotide sequence of SEQ ID NO:15; exon 5 comprises a polynucleotide sequence of SEQ ID NO:17; intron 2 comprises a polynucleotide sequence of SEQ ID NO:55; and intron 4 comprises a polynucleotide sequence of SEQ ID NO:

39. The primary transcript further comprises at least one exon 3a or a portion thereof of a Noctuidae hermaphroditic gene, and the exon 3a comprises the polynucleotide sequence of SEQ ID NO:

12. The primary transcript further comprises at least one exon 4b or a portion thereof of a Noctuidae hermaphroditic gene, and the exon 4b comprises the polynucleotide sequence of SEQ ID NO:14, and The primary transcript further comprises at least one intron 3 or a portion thereof of a Noctuidae hermaphroditic gene, and the intron 3 comprises the polynucleotide sequence of SEQ ID NO:

58.

33. The method according to claim 1, wherein, The promoter is the Hsp70 promoter, β-tubulin promoter, Hsp83 promoter, protamine promoter, actin promoter, Hsp70 minimal promoter, P minimal promoter, CMV minimal promoter, Acf5C-based minimal promoter, TRE3G promoter, BmA3 promoter fragment, or Adh core promoter.

34. The method of claim 33, wherein the promoter has a polynucleotide sequence of SEQ ID NO:18, SEQ ID NO:41, SEQ ID NO:63 or SEQ ID NO:

65.

35. The method according to any one of claims 1 to 34, wherein the expression system further comprises a transcriptional control element that controls transcription by the presence or absence of a chemical ligand.

36. The method of claim 35, wherein the transcriptional control element is a tetracycline-responsive element.

37. The method of claim 36, wherein the tetracycline-responsive element is tetOx1, tetOx2, tetOx3, tetOx4, tetOx5, tetOx6, tetOx7, tetOx8, tetOx9, tetOx10, tetOx11, tetOx12, tetOx13, tetOx14, tetOx15, tetOx16, tetOx17, tetOx18, tetOx19, tetOx20, or tetOx21.

38. The method according to claim 1, wherein the hermaphroditic gene of the Noctuidae family is derived from species of the genera *Spodoptera*, *Helicoverpa*, *Chrysodeixis*, *Anticarsia*, *Peridroma*, or *Heliothis*.

39. The method according to claim 38, wherein, The hermaphroditic genes of the Noctuidae family are derived from the fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), gray-winged armyworm (Spodopteralittoralis), cotton bollworm (Helicoverpa armigera), saucia armyworm (Peridroma saucia), cereal armyworm (Helicoverpa zea), silver armyworm (Chrysodeixis includens), pear bean armyworm (Anticarsiagemmatalis), or tobacco armyworm (Heliothis virescens).

40. A polynucleotide comprising the polynucleotide sequence of SEQ ID NO:

86.

41. A polynucleotide comprising the polynucleotide sequence of SEQ ID NO:

87.

42. A polynucleotide containing the polynucleotide sequence of SEQ ID NO:88.

Images