Transgenic safe sites in the genome of Bactrocera dorsalis and their screening methods and applications

Abstract

The present invention discloses a safe transgenic site of the citrus fruit fly genome and a screening method and application thereof. The present invention develops an Actin gene promoter, and further constructs two different citrus fruit fly strains from it, and finds a feasible safe transgenic insertion site of the citrus fruit fly genome, thereby providing important technical support for the strain development of the citrus fruit fly and the prevention and control work with gene editing as the core.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, and in particular to transgenic safety sites in the genome of the citrus fruit fly, and a screening method and application thereof. Background Art

[0002] Transgenic technology refers to a technical means of inserting exogenous gene fragments into the genome of an organism through a series of measures, which can enable transgenic organisms to acquire new genetic characteristics. At present, transgenic technology has been widely used in gene function research, variety improvement, and biosynthesis. In transgenic research, in order to avoid the impact of the introduction of exogenous genes on the host's own growth activity and phenotype, scientists have proposed the concept of safe sites (Genomic Safe Harbors, GSHs). It refers to the region in the genome that can maintain transgenic expression without significantly affecting the function of host cells. It should not overlap with any known functional sequence in the genome or affect the expression of nearby genes. GSHs play an important role in improving the effectiveness and safety of genome engineering, and also provide a reference for scientists and regulatory agencies to evaluate the safety and environmental impact of genetically modified organisms. Therefore, the identification and development of GSHs are very necessary for the construction of stable transgenic strains.

[0003] Bactrocera dorsalis belongs to the order Diptera, family Tetriphitidae, and is a common fruit and vegetable pest. It is mainly distributed in tropical and subtropical regions of Asia, harming more than 150 kinds of fruits and vegetables. In terms of transgenic technology, although transgenic technology based on the transposon piggybac has been successfully applied to Bactrocera dorsalis, which has played an important role in the study of gene function of Bactrocera dorsalis and the development of pest control technology, there is currently no report on GSHs of Bactrocera dorsalis. The identification of GSHs will lay the foundation for the study of transgenic technology of Bactrocera dorsalis. Summary of the invention

[0004] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide a safe transgenic site in the genome of the citrus fruit fly and its screening method and application. The present invention obtains an Actin gene promoter, and successfully constructs two different citrus fruit fly strains based on the promoter, and in this process, further explores and discovers multiple safe sites that can be used for transgenics, thereby providing important technical support for the strain development of the citrus fruit fly and the prevention and control work centered on gene editing methods.

[0005] The first aspect of the present invention provides an Actin gene promoter, wherein the Actin gene promoter comprises:

[0006] (1) an Actin gene promoter having a nucleotide sequence as shown in SEQ ID NO: 3;

[0007] (2) An Actin gene promoter variant comprising the nucleotide sequence shown in SEQ ID NO: 3 and having at least 90% homology, wherein the Actin gene promoter variant has a driving activity similar to that of (1).

[0008] In some embodiments of the present invention, the Actin gene promoter includes: an Actin gene promoter variant containing the nucleotide sequence shown in SEQ ID NO: 3 and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

[0009] In the present invention, the terms "similar" and "close" refer to variants having a driving activity that is at most 20% different from the driving activity of the Actin gene promoter in (1). In some embodiments of the present invention, the difference may be 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

[0010] The second aspect of the present invention provides a product containing the Actin gene promoter described in the first aspect of the present invention, the product comprising:

[0011] (1) An expression vector containing the Actin gene promoter according to claim 1;

[0012] (2) A transformant containing the Actin gene promoter according to claim 1;

[0013] (3) introducing a transformant containing the expression vector described in (1);

[0014] In some embodiments of the present invention, the expression vector includes a plasmid, a virus and a bacteriophage.

[0015] In some embodiments of the present invention, the transformants include microorganisms, plant cells and animal cells.

[0016] In some embodiments of the invention, the product does not involve animal or plant propagation material.

[0017] In some embodiments of the invention, the product comprises:

[0018] (1) the plasmid shown in SEQ ID NO: 4; or

[0019] (2) A derivative of (1) with the addition of non-functional sequences or groups.

[0020] In some embodiments of the present invention, the non-functional sequence includes a tag sequence, a fluorescent protein sequence and a resistance sequence.

[0021] In some embodiments of the invention, the group comprises a reporter group.

[0022] In some embodiments of the present invention, the reporter group includes a chromogenic enzyme, a fluorescent labeling group, a chemiluminescent labeling group, an isotope or a magnetic functional group.

[0023] In some embodiments of the present invention, the color developing enzyme may include peroxidase and alkaline phosphatase.

[0024] In some embodiments of the present invention, the fluorescent labeling group may include fluorescent proteins, rhodamines, fluoresceins, cyanine dyes, cyanine dyes, AlexaFluor dyes and / or quantum dot fluorescent groups.

[0025] In some embodiments of the present invention, the magnetic functional group may include a group capable of magnetic resonance imaging and changing relaxation efficiency.

[0026] In some embodiments of the invention, the isotope comprises a radionuclide.

[0027] The third aspect of the present invention provides the use of the Actin gene promoter described in the first aspect of the present invention or the product described in the second aspect in gene editing.

[0028] A fourth aspect of the present invention provides a gene editing method, comprising at least one of the following steps:

[0029] Use the Actin gene promoter described in the first aspect of the present invention to construct an expression element; or

[0030] The product described in the second aspect of the present invention is used for gene editing.

[0031] In the present invention, the term "gene editing" generally refers to the process of modifying a specific target of the genome of an organism through gene editing technology, including but not limited to any technical means to achieve the insertion, deletion or replacement of a specific gene.

[0032] The fifth aspect of the present invention provides a safe insertion site for a transgenic plant in the genome of the fruit fly. The safe insertion site for a transgenic plant in the genome of the fruit fly is located on the sequence shown in SEQ ID NO: 16 or 17.

[0033] In some embodiments of the present invention, the safe insertion site of the transgene in the Bactrocera dorsalis genome has TTAA specificity, which conforms to the transposition characteristics of piggyBac.

[0034] In some embodiments of the present invention, the location of the TTAA is as follows:

[0035] The A3-DsRed.1 insertion site is located on chromosome 5 (5'-TTGACTGGATGGGCATGATT TTAA TGAGCCAGCGAATAACTTAT-3' (SEQ ID NO: 16));

[0036] The A3-DsRed.2 insertion site is located on chromosome 4 (5'-CCAAAAATATCACTTTTATT TTAA TAAACACGTTTAATTTTTAA-3' (SEQ ID NO: 17)).

[0037] The sixth aspect of the present invention provides a method for obtaining a safe transgenic insertion site in the genome of Bactrocera dorsalis according to the fourth aspect of the present invention, comprising the following steps:

[0038] The transgenic strain larvae constructed using the Actin gene promoter described in the first aspect of the present invention or the product described in the second aspect are obtained, and the sequencing data are quality controlled and filtered after removing the linker, and then the data after quality control filtering are compared with the reference sequence file to obtain a bam file; statistical screening is performed in the bam file to predict candidate transgenic safe insertion sites, and the candidate transgenic safe insertion sites are visualized using IGV, and available transgenic safe insertion sites of the citrus fruit fly genome are obtained according to the breakpoints.

[0039] In some embodiments of the present invention, the reference sequence file is obtained by merging the reference genome sequence file of Bactrocera dorsalis and the constructed transgenic plasmid sequence file.

[0040] In some embodiments of the invention, the quality control filtering is used to remove low-quality reads.

[0041] In some embodiments of the present invention, the comparison between the data after quality control filtering and the reference sequence file is performed using BWA software. Of course, those skilled in the art can use other software for comparison processing according to actual use requirements.

[0042] In some embodiments of the present invention, the statistics and screening of the bam files are performed using samtools software. Of course, those skilled in the art can use other software to perform statistics and screening according to actual use requirements.

[0043] In some embodiments of the present invention, the screening requirement is: pair-end reads with one end aligned to the Bactrocera dorsalis reference genome and the other end aligned to the vector sequence.

[0044] In some embodiments of the invention, the method further comprises PCR verification.

[0045] In some embodiments of the present invention, the primers used for PCR verification are shown in Table 3.

[0046] The sixth aspect of the present invention provides the application of the safe transgenic insertion site of the citrus fruit fly genome described in the fourth aspect of the present invention in gene editing.

[0047] In some embodiments of the present invention, the object of gene editing is Tephritidae insects.

[0048] In some embodiments of the present invention, the object of gene editing is Bactrocera dorsalis.

[0049] A seventh aspect of the present invention provides a gene editing method for Tephritidae insects, comprising:

[0050] The target sequence is transferred into the fourth safe transgenic insertion site of the Bactrocera dorsalis genome of the present invention.

[0051] The beneficial effects of the present invention are:

[0052] 1. BdorActinA3-1 in the present invention P-2k The promoter can drive broad and strong expression of fluorescent markers, and can be used in the later stage for overexpression studies of important genes in transgenics.

[0053] 2. BdorActinA3-1 in the present invention P-2k The promoter can be used as a screening marker in transgenic construction to screen strains at any time, thereby improving screening efficiency.

[0054] 3. The two transgenic strains constructed in the present invention survive normally and are easy to propagate in the laboratory. The two identified insertion sites can be used as transgenic safe sites (Genomic Safe Harbors, GSHs) on the genome. They will provide support for the study of phenotypes (electrophysiology, olfactory behavior, etc.) that are of concern in the development of citrus fruit fly attractants, and will help to apply transgenic genetic manipulation technology to the development of behavioral regulators in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Schematic diagram of the original map of plasmid pBac[3×P3-DsRedaf]

[0056] Figure 2 The results of BdorActin family analysis and BdorActinA3a-1 promoter region prediction are shown in Figure 1. A is the phylogenetic tree of candidate BdorActins and reported Diptera Actins; B is the expression pattern of BdorActins at different developmental stages; E represents eggs, and E1 to E6 represent eggs at different time intervals after egg laying, which are 30 minutes, 3 hours, 6 hours, 18 hours, 24 hours, and 30 hours, respectively; L represents larvae, and L1 to L3 correspond to larvae of different ages, including newly eclosing larvae, 2nd or 3rd instar larvae, and mature larvae approaching the pupation stage; P represents pupae, and P1 to P4 refer to pupae of different ages, including initial pupae, 3-day-old, 6-day-old, and pupae about to eclode; A represents adults, and the data were downloaded from the GenBank database (sequence accession numbers: SRR9945663, SRR9945664, and SRR9945668); C is the prediction result of the BdorActin-A3a-1 promoter sequence.

[0057] Figure 3 The construction process of transgenic lines. A is a schematic diagram of embryo injection; B is the hybridization process of transgenic lines; C is the BdorActinA3a-1 P-2k -Dsred1 (i.e., A3-DsRed.1) expression patterns at various developmental stages, including eggs, embryos, larvae, pupae, and adults.

[0058] Figure 4 BdorActinA3a-1 P-2k -Expression of Dsred1 in different tissues. A is BdorActinA3a-1 P-2k -Expression of Dsred1 in antennae; B is BdorActinA3a-1 P-2k -Expression of Dsred1 in mouthparts; C is BdorActinA3a-1 P-2k -Expression of Dsred1 in the mandibular palp; D stands for BdorActinA3a-1 P-2k -Expression of Dsred1 in the forefoot; E is BdorActinA3a-1 P-2k -Dsred1 expression in the midfoot; F is BdorActinA3a-1 P-2k -Dsred1 expression in the hind legs; G stands for BdorActinA3a-1 P-2k -Expression of Dsred1 in the brain; H stands for BdorActinA3a-1 P-2k -Expression of Dsred1 in male genitalia; I is BdorActinA3a-1P-2k -Expression of Dsred1 in female genitalia.

[0059] Figure 5 Insertion site and PCR verification of transgenic lines.

[0060] Figure 6 Comparison of survival index between wild type and transgenic strains. A is the comparison of hatching rate between wild type and transgenic strains; B is the comparison of eclosion rate between wild type and transgenic strains; C and D are the adult survival curves of wild type and transgenic strains, respectively. The figure corresponds to the survival index of males and females from 0 to 50 days after eclosion, and the data are expressed as mean ± standard error. *p<0.05, **p<0.01, ****p<0.0001; ns indicates no significant difference.

[0061] Figure 7 Comparison of important olfactory behaviors between wild type and transgenic strains. A is the comparison of male tropism of wild type and transgenic strains to ME; B is the comparison of female tropism of wild type and transgenic strains to TMP; C is the trend of male wing flapping behavior of wild type and transgenic strains, with 5-minute intervals (0 minutes represent 16:30, 330 minutes represent 22:00); D is the comparison of mating behavior between wild type and transgenic strains, and the number of matings is counted every 10 minutes (0 minutes represent 16:30, 330 minutes represent 22:00).

[0062] Figure 8 The electrophysiological responses of wild type and transgenic strains to important compounds, among which A is a schematic diagram of antennal potential (EAG) response of Bactrocera dorsalis to compounds; B is a comparison of electrophysiological responses of male insects caused by 23 compounds; C is a comparison of electrophysiological responses of female insects caused by 23 compounds. Data are expressed as mean ± standard error. ns indicates no significant difference. DETAILED DESCRIPTION

[0063] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0064] Screening of Actin gene expressed in ubiquitously tissues and construction of transgenic plasmid

[0065] (1) Annotation, phylogenetic analysis and structure prediction of the BdorActin family:

[0066] The inventors collected the Actin gene protein data of dipteran insects published by NCBI, constructed a hidden Markov model (HMM) using Hmmer (refer to Potter et al. 2018), and used this model to search the protein sequence of the Bactrocera dorsalis genome (National Center for Bioinformatics GSA database, PRJCA020830), screening for evalue < 1e -5 The results were used as gene set 1. The protein sequences of the Actin gene of Drosophila melanogaster were collected from the Swiss-prot database and multiple alignments were performed with the protein sequences of the Bactrocera dorsalis genome using Psi-blast (refer to Thangavel et al. 2023). -5 The result was taken as gene set 2. The intersection of the two gene sets was selected, and the amino acid sequences of the genes in them were verified by domain structure in the interpro (https: / / www.ebi.ac.uk / interpro / ) database, and the Actin candidate gene set was screened based on the Actin domain in the amino acid sequence.

[0067] The online database Uniprot (https: / / www.uniprot.org / ) was used to download the Actin amino acid sequences of five Diptera species, Bactrocera minax, Bactrocera oleae, Rhagoletis pomonella, Rhagoletis zephyria, and Drosophilamelanogaster. The amino acid sequences in the Actin candidate gene set of Bactrocera dorsalis obtained above were used for multiple sequence alignment using MAFFT (v 7.515) (method reference Katoh et al. 2013). The ModelFinder option of IQ-TREE (v 2.2.0.3) (method reference Minh et al. 2020) software was used to select the optimal nucleotide substitution model, and the phylogenetic tree was constructed using the maximum likelihood method.The accession numbers of the actin sequences of five Diptera species, including Bactroceraminax, Bactrocera oleae, Rhagoletis pomonella, Rhagoletis zephyria, and Drosophilamelanogaster, are as follows: QIZ85986.1 (Bactrocera minaxactin), XP 014098863.1 (Bactrocera oleae actin, cytoplasmic A3a), XP 036343282.1 (Rhagoletis pomonella actin, indirect flight muscle-like), XP 036318112.1 (Rhagoletis pomonella actin, larval muscle), XP 017492682.1 (Rhagoletis zephyriaactin, clone 302-like), XP 017480986.1 (Rhagoletis zephyriaactin, clone 302-like), XP 017481167 ... actin, larvalmuscle), actin 79B), NP523625.1 (Drosophila melanogaster actin 42A) and NP 001014725.1 (Drosophila melanogaster actin 5C).

[0068] The genome map (National Center for Bioinformation GSA database, PRJCA020830) was further used in combination with the transcriptome data of the genome assembly (National Center for Bioinformation GSA database, PRJCA020830). The Actin gene in the candidate gene set was aligned to the reference genome using Blat (see Kent, 2002) to obtain its location information on the genome. Samtools (see Li et al. 2009) was used to intercept the 10K upstream and downstream regions of the Actin gene to obtain the complete nucleic acid sequence of the region. Hisat2 (v 2.2.1) (see Kim et al. 2015) was then used to align the transcriptome data to the intercepted genome region, and the transcript reads aligned to the target genome region were assembled using Stringtie (see Pertea et al. 2015) to obtain the candidate transcript structure. Transdecoder (refer to Haas et al. 2013) was used to predict the transcript open reading frame (ORF), and the aligned genomic regions, transcriptomic evidence, transcript reads coverage, and predicted ORFs were imported into IGV (refer to Robinson et al. 2023) for IGV visualization. The credible gene structure was comprehensively judged based on the genomic location information and transcriptomic evidence of the candidate Actin gene.

[0069] (2) Analysis of the expression pattern of BdorActins at different developmental stages:

[0070] Transcriptome data (sequencing) of each developmental stage (egg, larvae and pupa) of B. dorsalis and adult transcriptome data reported in the GenBank database (accession numbers: SRR9945663, SRR9945664, SRR9945668) were collected to assemble transcripts and map them to the reference genome. Stringtie was then used to calculate the expression level and TPM value, and the original expression matrix and TPM value matrix were integrated. pheatmap (see Hu, 2021) was used in R to construct the expression profile of the egg, larvae, pupa and adult developmental periods for the B. dorsalis Actin candidate gene.

[0071] (3) Prediction, cloning and transgenic plasmid construction of BdorActin promoter sequence:

[0072] According to the predicted BdorActins structure in the above steps, the start codon position of the target Actin was determined in the genome. The 2k region upstream of the start codon was selected as the candidate promoter region of the gene. The promoter region was cloned using gDNA (Actin-A3a-1-F: 5'-TGTCGCTTTGTGA ACTAGAGAAACA-3' (SEQ ID NO: 1) and Actin-A3a-1-R: 5'-TTTGTTTTCTGAAAACAAAAA-3' (SEQ ID NO: 2)) as a template. The plasmid [pbac-3×p3-dsred-attp] was used as a transgenic vector (i.e., pBac [3×P3-DsRedaf] in Horn et al. 2002). The plasmid map is shown in Figure 1 As shown, 3×p3 was replaced with the cloned promoter region using conventional methods in the art to synthesize the transgenic plasmid to be constructed. The plasmid was transformed into DH5α competent cells for sequence verification and used Plasmids were extracted using Plasmid Plus Midi Kit (QIAGEN, Germany). After sequence confirmation, plasmids were purified using phenol-chloroform and used for embryo injection.

[0073] In this example, a total of 6 candidate BdorActins were obtained (as shown in Table 1).

[0074] Table 1 Candidate Actin genes of Bactrocera dorsalis

[0075]

[0076] Phylogenetic tree Figure 2 A shows that these BdorActins are clustered with homologous sequences of Tephritid and D. melanogaster. Through the transcriptome data of the developmental period, the inventors screened out a gene BdorActin-A3a-1 ( Figure 2 B). The inventors believe that the promoter of BdorActinA3a-1 may have broad and efficient driving activity.

[0077] Then, the inventors predicted the promoter region of BdorActinA3a-1 through genomic data and intercepted the 2k sequence BdorActinA3a-1P-2k upstream of the transcription start site (eg Figure 2 C, the nucleotide sequence is shown in SEQ ID NO: 3), and the transgenic plasmid pBac[BdorActinA3a-1P-2k-Dsred1; attP] was constructed (the flowchart is shown in Figure 3 A and 3B, and the nucleotide sequence is shown in SEQ ID NO: 4).

[0078] Construction of transgenic strains of Bactrocera dorsalis

[0079] (1) In vitro synthesis of transposase Hybase mRNA to improve transposition efficiency:

[0080] The specific synthesis steps are as follows: first, the T7-hybase transcription template was amplified from the pTD1-T7-hybase plasmid (nucleotide sequence as shown in SEQ ID NO: 5) by designing specific primers (primers: pTD1-T7-F: 5'-ATGCGGTGTGAAATACCGCACAGATGCG-3' (SEQ ID NO: 6); pTD1-T7-R: 5'-TAGAGGCCCCAAGGGGTTATGCTAG-3' (SEQ ID NO: 7)). Then, the Hybase mRNA was transcribed in vitro using mMESSAGE mMACHINETM T7 (Thermo Fisher Scientific). The transcription product (mRNA) was purified by phenol-chloroform and stored in a -80°C refrigerator for future use.

[0081] Embryo collection and microinjection were performed according to the method reported by Yuan (2022). The working concentration of transgenic plasmid and Hybase mRNA was approximately 300 ng / μL. After thorough mixing, the FemtoJet and InjectMan 4 microinjection systems (Eppendorf) were used for injection. The injected embryos were incubated for about 1 day at a temperature of (26±1)°C and a relative humidity of (60±5)%. The hatched larvae were selected and raised on larval feed. All G0 adults were mated individually with the wild type. G1 pupae were screened for the presence of DsRed1 expression. Transgenic positive individuals were selected for backcrossing with wild-type partners. The transgenic lines were cultivated to homozygous by selecting larvae or pupae with bright fluorescence. During the selection process, the expression of fluorescent protein DsRed1 was detected using an Olympus stereo microscope BX51 (Olympus), which was equipped with a RED bandpass filter (with excitation filter BP540-550, dichroic beam splitter DM570 and barner filter BA575-625). Images were captured using an Olympus image acquisition system.

[0082] The results are shown in Table 2.

[0083] Table 2 Microinjection and screening records

[0084]

[0085] In this example, a total of 600 eggs were injected and 95 G0 individuals were obtained. After hybridization with the wild type, as shown in Table 2, 11 positive individuals expressing DsRed1 were obtained in the G1 generation. Through breeding and screening, two strains with good growth conditions were finally retained and named A3-DsRed.1 and A3-DsRed.2. Obvious expression of DsRed1 was observed in the embryos, larvae, pupae and adults of these two strains ( Figure 3 C). Further verification revealed that A3-DsRed.2 had the same expression pattern.

[0086] The inventors further evaluated the expression of the transgenic elements in the olfactory organs and their effects on typical olfactory behaviors and antennal electrophysiological responses.

[0087] The results showed that the antennae, mouthparts, mandibular palps, front, middle and hind legs, external genitalia and brain of male and female insects all showed strong DsRed1 expression ( Figure 4 ). Further verification revealed that A3-DsRed.2 had the same expression pattern.

[0088] Confirmation of transgene insertion site

[0089] Larvae were collected from the obtained transgenic lines for resequencing. The original resequencing data were processed by trimmomatic software to remove adapters and filter low-quality reads to obtain high-quality second-generation sequencing pair-end reads. The reference genome sequence file of B. dorsalis and the transgenic plasmid sequence file were merged into a new reference sequence file. The high-quality second-generation sequencing pair-end reads were aligned with the new reference sequence file using BWA software to obtain a bam file containing the alignment. The bam file generated by the alignment was statistically analyzed using samtools software to screen pair-end reads that aligned to the reference genome of B. dorsalis at one end and to the vector sequence at the other end. The suspected insertion sites in the genome of B. dorsalis were predicted based on the alignment positions. The new reference sequence file and the bam file generated by the alignment were imported into IGV to visualize the alignment of the pair-end reads of the suspected insertion sites. The reliable insertion site position was determined based on whether there were consistent breakpoints in the pair-end reads of the suspected insertion site position. Subsequently, primers for PCR verification were designed based on the insertion site.

[0090] The primers are shown in Table 3.

[0091] Table 3 Primer information

[0092]

[0093] The results showed that through PCR verification of heavy genome sequencing, the inventors determined the insertion position of the transgenic elements in the two strains. The A3-DsRed.1 insertion site was located on chromosome 5. The A3-DsRed.2 insertion site is located on chromosome 4 Both sites are TTAA-specific (underlined and bold), consistent with the translocation properties of piggyBac ( Figure 5 ). These results indicate that we have successfully achieved germline transformation of Bactrocera dorsalis using piggyBac.

[0094] Determination of the survival index of transgenic lines

[0095] The following parameters were measured for the transgenic lines obtained in the above examples:

[0096] (1) Hatching rate: Mix 12-day-old virgin male and female worms (12 each), and collect fresh eggs using an artificial orange juice tray at 16 days of age after sufficient mating. Randomly select 30 eggs and place them in a culture dish with a damp black cloth. Place the culture dish in an incubator and adjust the temperature to (26±1)℃ and relative humidity to (60±5)%. The black cloth must be kept moist throughout the process. Count the number of eggs that finally hatch.

[0097] (2)Emergence rate and adult growth curve:

[0098] Collect enough offspring from the above fully mated females. Randomly select 30 healthy pupae and record their emergence rate. At the same time, observe the survival curve of adults until the 50th day of adulthood. The experimental environment conditions are temperature (26±1)℃ and relative humidity (60±5)%.

[0099] In order to evaluate the effect of transgenic on the key survival parameters of Bactrocera dorsalis, the inventors compared the differences between wild-type and transgenic strains in hatching rate, eclosion rate and adult survival curve through the above experiment. The results showed that the egg hatching rate of transgenic strains was no different from that of wild-type, both above 83% (A3-DsRed.1: P = 0.4458; A3-DsRed.2: P = 0.0983) ( Figure 6 A). The eclosion rate decreased slightly (A3-DsRed.1: P = 0.0038; A3-DsRed.2: P = 0.0145), the eclosion rate of the wild type was higher than 90%, and the eclosion rate of the two transgenic lines was also close to 80% ( Figure 6B). There was no significant difference in the survival rate of wild-type and transgenic adult insects (A3-DsRed.1: P(male) = 0.3683, P(female) = 0.4176; A3-DsRed.2: P(male) = 0.2594, P(female) = 0.7472). Among males, 90% could survive for more than 20 days, and 77.5% could survive for up to 50 days ( Figure 6 C). Among females, 90% can survive for more than 20 days, and 80% can survive up to 50 days ( Figure 6 D) In ​​summary, it can be concluded that the transgenic lines mediated by piggyBac are easy to maintain and can be successfully propagated in the laboratory. They are able to maintain stable genetics, making them effective for future experiments.

[0100] Observation of typical behaviors of transgenic lines

[0101] (1) Preference for attractants:

[0102] In this example, the tendency behavior of methyl eugenol (ME) and 2,3,5-trimethylpyrazine (TMP) was tested: the above attractants were diluted to 100 μg / μL in paraffin oil. The tendency rate was tested using artificial traps, which were transparent conical bottles of specific dimensions. Each experimental cage contained two trap bottles: one with the test odor ME or TMP and the other with paraffin oil as a control. Adult males or females were placed in cages equipped with these traps and their preference for the attractants was observed.

[0103] The experimental time of ME was 9:00-10:00 AM, and the dosage of ME was 1 mg. The experimental time of TMP was 4:30-10:00 PM, and the dosage of TMP was 10 mg.

[0104] (2) Wing flapping behavior:

[0105] The wing flapping behavior was recorded by an automatic video recording device. The system consists of a camera connected to a computer, a light board with adjustable light intensity, and a behavior cage. The insects used in the experiment were 12-day-old unmated male insects. The vibration of the wings of male adults accompanied by anal beating was defined as typical wing-flapping behavior. The number of males performing wing flapping was counted every 5 minutes. A total of 12 male insects were recorded in each experiment. The frequency of wing flapping directly represents the number of wing flappings performed by the male. For example, if 5 males started wing flapping behavior within 5 minutes, the frequency of wing flapping was defined as 5. The experimental temperature was 26±1℃, the humidity was (60±10)%, and the experimental time was 16:30-22:00.

[0106] (3) Mating behavior:

[0107] The experimental apparatus and conditions were the same as those in the above embodiment. The insects used in the experiment were 12-day unmated male and female insects. The typical mating behavior was that the male insect jumped onto the back of the female insect and flapped its wings. After the sexual organs were successfully connected, stable mating was achieved, and this process usually lasted for more than 2 hours. Each experiment recorded 12 pairs of 12-day unmated male and female insects. The experimental temperature was 26±1°C, the humidity was (60±10)%, and the experimental time was 16:30-22:00. The number of matings was counted every 10 minutes.

[0108] The results showed that the transgenic males had no difference in response to methyl eugenol compared with the wild type (A3-DsRed.1: P = 0.9866; A3-DsRed.2: P = 0.7887) ( Figure 7 A), but the response of females to TMP was slightly decreased (A3-DsRed.1: P = 0.0007; A3-DsRed.2: P = 0.0003) ( Figure 7 B). ME was able to attract more than 51.33% of transgenic males, which was consistent with the response characteristics of the wild type; TMP attracted about 54% of wild type females, and the attraction rate of the transgenic strain was close to 38%. Wing flapping is a signature courtship behavior of the orange fruit fly before mating, which was significantly affected by transgenic insertion. Compared with the wild type, the courtship behavior of the transgenic strain in the first 200 minutes was significantly delayed ( Figure 7 C). Mating behavior was also affected by the transgenic elements. About 53% of the transgenic adults (A3-DsRed.1: 55%; A3-DsRed.2: 53.3%) began to mate at the end of the experiment, which was significantly lower than the 96.7% mating ratio of the wild type ( Figure 7 D).

[0109] Measurement of antennal electrophysiological responses of transgenic lines

[0110] The measurement was performed by EAG recording (see Xu et al. 2020). The head of the male fruit fly was removed, and one end of the antenna was cut off. Two glass electrodes were prepared and filled with 0.1M KCl solution. The reference electrode was connected to the head cut, and the recording electrode was connected to the end of the antenna cut. Recording was performed under a BX 51 microscope (Olympus).

[0111] According to previous studies (Zhang et al, 2023), compounds with obvious EAG response values ​​(>0.2mV) were selected, and a total of 23 compounds were selected. All compounds were diluted in paraffin oil, and the dose for each test was 100μg, with paraffin oil as the control. During the experiment, the odor source was dripped onto the filter paper, placed in a Pasteur pipette, and sealed. The controller (CS-55, Syntech) was used for airflow stimulation. The stimulation airflow was set to 10mL / s, the stimulation duration was 0.4s, and the interval between stimulations was about 30 seconds to ensure the activity of the antennae. The signal was collected using a universal probe preamplifier, and then the signal was converted by a digital-to-analog converter (IDAC-4-USB, Syntech). The collected signal was analyzed using EAG pro 2.0. The EAG reaction signal is the signal of the compound to be tested minus the signal of paraffin oil.

[0112] The results showed that there were no differences in the electrophysiological responses of the transgenic strains and the wild type to 23 important compounds, including esters, alcohols, aldehydes, terpenes and others. Figure 8 ). These results confirm the successful expression of the reporter gene in the olfactory organ and suggest that although transgenic modifications do not alter general electrophysiological responses, they can affect specific olfactory and reproductive behaviors of B. dorsalis.

[0113] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

[0114] References

[0115] Haas B J,Papanicolaou A,Yassour M,Grabherr M,Blood P D,Bowden J,Couger M B,Eccles D,Li B,Lieber M,MacManes M D,Ott M,Orvis J,Pochet N,StrozziF,Weeks N,Westerman R,William T,Dewey C N,Henschel R,LeDuc R D,Friedman N,Regev A.2013.De novo transcript sequence reconstruction fromRNA-seq using theTrinity platform for reference generation and analysis.Nature protocols,8,1494–1512.Horn C,Schmid BG,Pogoda F S,Wimmer E A.2002.Fluorescenttransformation markers for insect transgenesis.

[0116] Insect Biochemistry and Molecular Biology,32,1221–1235.

[0117] Katoh K,Standley D M.2013.MAFFT multiple sequence alignment softwareversion 7:improvements inperformance and usability.Molecular biology andevolution,30,772–780.

[0118] Kent W J.2002.BLAT--the BLAST-like alignment tool.Genome research,12,656–664.

[0119] Kim D,Langmead B,Salzberg S L.2015.HISAT:a fast spliced aligner withlow memory requirements.Naturemethods,12,357–360.

[0120] Li H,Durbin R.2009.Fast and accurate short read alignment withBurrows-Wheeler transform.Bioinformatics

[0121] (Oxford,England),25,1754–1760.

[0122] Minh B Q,Schmidt H A,Chernomor O,Schrempf D,Woodhams M D,von HaeselerA,Lanfear R.2020.

[0123] IQ-TREE 2:New Models and Efficient Methods for Phylogenetic Inferencein the Genomic Era.Molecularbiology and evolution,37,1530–1534.

[0124] Pertea M,Pertea G M.,Antonescu C M,Chang T C,Mendell J T,Salzberg SL.2015.StringTie enables improvedreconstruction of a transcriptome from RNA-seq reads.Nature biotechnology,33,290–295.

[0125] Potter S C,Luciani A,Eddy S R,Park Y,Lopez R,Finn R D.2018.HMMER webserver:2018update.NucleicAcids Research,46,W200-W204.

[0126] Robinson J T,Thorvaldsdottir H,Turner D,Mesirov J P.2023.igv.js:anembeddable JavaScript implementationof the Integrative Genomics Viewer(IGV).Bioinformatics(Oxford,England),39,btac830.

[0127] Thangavel G,Hofstatter P G,Mercier R,Marques A.2023.Tracing theevolution of the plant meiotic molecularmachinery.Plant Reproduction,36,73-95.

[0128] Xu J,Liu W,Yang D H,Chen S Q,Chen K,Liu Z L,Yang X,Meng J,Zhu G H,Dong S L,Zhang Y,Zhan S,Wang G R,Huang Y P.2020.Regulation of olfactory-basedsex behaviors in the silkworm by genes in thesex-determination cascade.PLoSGenetics,16,e1008622.

[0129] Yuan J,Zhang J,Zhang Y,Wuyun Q Q G,Liu W,Yan S C,Wang GR.2022.Protocols for CRISPR / Cas9

[0130] Mutagenesis of the Oriental Fruit Fly Bactrocera dorsalis.Jove-journal of Visualized Experiments,(187).

[0131] PMID:36279540.Available from: https: / / doi.org / 10.3791 / 64195.

[0132] Zhang Y,Wuyun Q Q G,Wang Q,Luo Z C,Yuan J X,Zhang J,Yan S C,Liu W,Wang G R.2023.MFStransporter Bdorwp does not affect antennalelectrophysiology but regulates reproductive behaviors inBactroceradorsalis.Journal of Agricultural and Food Chemistry,71,17014-17024.

Claims

1. An Actin gene promoter, characterized in that: The Actin gene promoter is: the Actin gene promoter with the nucleotide sequence shown in SEQ ID NO:

3.

2. A product containing the Actin gene promoter according to claim 1, characterized in that: The products include: (1) An expression vector containing the Actin gene promoter according to claim 1; (2) A transformant containing the Actin gene promoter according to claim 1; or (3) introducing a transformant containing the expression vector described in (1); The expression vectors include plasmids, viruses and bacteriophages; The transformants include microorganisms, plant cells and animal cells; The products described do not involve animal or plant propagation materials.

3. The product according to claim 2, characterized in that The products include: (1) the plasmid shown in SEQ ID NO: 4; or (2) a plasmid as shown in SEQ ID NO: 4, to which a non-functional sequence or a reporter group is added; The non-functional sequences include tag sequences, fluorescent protein sequences and resistance sequences.

4. Use of the Actin gene promoter according to claim 1 or the product according to any one of claims 2 to 3 in gene editing.

5. A gene editing method comprising at least one of the following steps: Using the Actin gene promoter described in claim 1 to construct an expression vector; or Use the product described in any one of claims 2-3 for gene editing.

6. A method for obtaining a safe transgenic insertion site in the genome of Bactrocera dorsalis, comprising the following steps: Obtain transgenic strain larvae constructed using the Actin gene promoter of claim 1 or the product of any one of claims 2-3, perform quality control filtering on the sequencing data after removing the linker, and then compare the quality control filtered data with the reference sequence file to obtain a bam file; perform statistical screening in the bam file to predict candidate transgenic safe insertion sites, use IGV to visualize the candidate transgenic safe insertion sites, and obtain available transgenic safe insertion sites in the citrus fruit fly genome according to the breakpoints; in, The reference sequence file is obtained by merging the reference genome sequence file of Bactrocera dorsalis and the constructed transgenic plasmid sequence file; The safe insertion site of the transgenic gene in the genome of the oriental fruit fly is A3-DsRed.1 or A3-DsRed.2; A3-DsRed.1 is located on SEQ ID NO: 16, and the sequence of SEQ ID NO: 16 is: 5'-ttgactggatgggcatgatt ttaa tgagccagcgaataacttat-3′, the insertion site A3-DsRed.1 is located at the TTAA sequence underlined in the above sequence; A3-DsRed.2 is located on SEQ ID NO: 17, and the sequence of SEQ ID NO: 17 is: 5'-ccaaaaatatcacttttatt ttaa taaacacgtttaatttttaa-3', the insertion site A3-DsRed.2 is located at the TTAA sequence underlined in the above sequence.

7. Application of safe transgenic insertion sites in the genome of Bactrocera dorsalis in gene editing; The safe insertion site of the transgenic gene in the genome of the oriental fruit fly is A3-DsRed.1 or A3-DsRed.2; A3-DsRed.1 is located on SEQ ID NO: 16, and the sequence of SEQ ID NO: 16 is: 5'-ttgactggatgggcatgatt ttaa tgagccagcgaataacttat-3′, the insertion site A3-DsRed.1 is located at the TTAA sequence underlined in the above sequence; A3-DsRed.2 is located on SEQ ID NO: 17, and the sequence of SEQ ID NO: 17 is: 5'-ccaaaaatatcacttttatt ttaa taaacacgtttaatttttaa-3', the insertion site A3-DsRed.2 is located at the TTAA sequence underlined in the above sequence.

8. The use according to claim 7, characterized in that: The object of the gene editing is Tephritidae insects.

9. A gene editing method for Tephritidae insects, comprising: The target sequence is transferred into the safe transgenic insertion site of the Bactrocera dorsalis genome; The safe insertion site of the transgenic gene in the genome of the oriental fruit fly is A3-DsRed.1 or A3-DsRed.2; A3-DsRed.1 is located on SEQ ID NO: 16, and the sequence of SEQ ID NO: 16 is: 5'-ttgactggatgggcatgatt ttaa tgagccagcgaataacttat-3′, the insertion site A3-DsRed.1 is located at the TTAA sequence underlined in the above sequence; A3-DsRed.2 is located on SEQ ID NO: 17, and the sequence of SEQ ID NO: 17 is: 5'-ccaaaaatatcacttttatt ttaa taaacacgtttaatttttaa-3', the insertion site A3-DsRed.2 is located at the TTAA sequence underlined in the above sequence.

Images