Nucleotide for controlling coleopteran pests and method therefor

Abstract

Provided is a method for controlling Monolepta hieroglyphica by means of using RNAi technology to reduce or silence the expression of a target gene combination in Monolepta hieroglyphica. The method selects MhCOPI gamma and Mhmi2 as target genes, designs a target sequence, and brings Monolepta hieroglyphica into contact with a corresponding dsRNA molecule, so as to inhibit the growth, development and reproduction of same and control the number of pests in the fields. Therefore, the control method is environment-friendly, efficient and safe.

Description

Nucleotides and methods for controlling coleopteran pests Technical Field

[0001] The present invention belongs to the field of agricultural biotechnology and relates to a nucleotide for controlling coleopteran pests and a method thereof, and in particular to a method for controlling the two-spotted firefly leaf beetle by reducing or shutting down the expression of a target gene combination in the leaf beetle using RNAi technology. Background Art

[0002] The two-spotted leaf beetle (Monolepta hieroglyphica (Motschulsky)) belongs to the order Coleoptera, family Chrysomelidae. Adults primarily infest corn leaves, feeding on the mesophyll, leaving irregular white, reticular spots and holes that severely impact photosynthesis. In August, they feed on the silk of the corn ear, disrupting pollination. They also feed on grain-filling kernels, causing ear rot. Severe infestations can cause widespread yield loss or even total crop failure.

[0003] Common control methods for the two-spotted leaf beetle include agricultural, biological, and chemical control. Agricultural control, such as weed removal and fertilization, requires significant human and material resources. Chemical control requires spraying pesticides, which can cause phytotoxicity and may not be entirely effective due to the beetle's wide range of infestations and high reproductive capacity. Biological control, however, requires the protection and utilization of natural enemies over a period of time and under certain conditions, and may not be able to rapidly control the pest population. Therefore, more economical, environmentally friendly, and effective methods are currently needed to control the two-spotted leaf beetle population.

[0004] RNAi technology has demonstrated significant advantages and application prospects in the control of agricultural pests. RNAi technology can design double-stranded RNA (dsRNA) that targets specific pests. Through genetic modification, plants can produce dsRNA that targets specific genes in insect tissues. These dsRNAs can specifically silence the pest's genes, leading to the insect's death, without affecting non-target organisms, including beneficial insects and humans. Therefore, RNAi technology has advantages such as high specificity, environmental friendliness, and reduced use of chemical pesticides. However, to date, all research using RNAi technology to control pests has only focused on direct lethality or growth inhibition of the current generation, or only on affecting the growth and development of the next generation, i.e., parental RNAi. These studies have resulted in poor or incomplete control effects, and can easily lead to resistance in the target pests. Once resistance develops, RNAi technology may lose its value in pest control.

[0005] Summary of the Invention

[0006] The present invention provides an isolated polynucleotide comprising at least one polynucleotide sequence selected from the group consisting of:

[0007] (a) the polynucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 22;

[0008] (b) a polynucleotide sequence having at least 87%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to at least 88, at least 156, at least 169, or at least 325 consecutive polynucleotides of SEQ ID NO: 1 or a polynucleotide sequence having at least 83% sequence identity to at least 144, at least 201, or at least 345 consecutive polynucleotides of SEQ ID NO: 22;

[0009] (c) any one of the polynucleotide sequences shown in SEQ ID NO: 2 to SEQ ID NO: 21 or any one of the polynucleotide sequences shown in SEQ ID NO: 23 to SEQ ID NO: 26;

[0010] (d) a polynucleotide sequence that hybridizes or is complementary to the polynucleotide sequence defined in any one of (a) to (c) above under stringent conditions.

[0011] The present invention also provides a polynucleotide composition comprising any one of the following polynucleotide sequences or their complementary sequences:

[0012] (a) the polynucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 22;

[0013] (b) a polynucleotide sequence having at least 87%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to at least 88, at least 156, at least 169, or at least 325 contiguous polynucleotides of SEQ ID NO: 1 and at least 83% sequence identity to at least 144, at least 201, or at least 345 contiguous polynucleotides of SEQ ID NO: 22;

[0014] (c) any one of the polynucleotide sequences shown in SEQ ID NO: 2 to SEQ ID NO: 21 and any one of the polynucleotide sequences shown in SEQ ID NO: 23 to SEQ ID NO: 25;

[0015] Preferably, the composition comprises the polynucleotides shown in SEQ ID NO: 2 and SEQ ID NO: 23 or their complementary sequences.

[0016] More preferably, the composition is the polynucleotide shown in SEQ ID NO: 26 or its complementary sequence.

[0017] The present invention also provides a polynucleotide encoding a double-stranded RNA molecule, comprising:

[0018] (1) any one of the polynucleotide sequences or polynucleotide composition sequences described above;

[0019] (2) spacer sequences; and

[0020] (3) The reverse complementary sequence of the polynucleotide sequence or polynucleotide composition sequence described in item (1);

[0021] Preferably, the spacer sequence is SEQ ID NO: 27.

[0022] The present invention also provides an expression cassette comprising the polynucleotide or the polynucleotide composition under the control of an operably linked regulatory sequence.

[0023] The present invention also provides a plant transformation vector comprising the polynucleotide or the polynucleotide composition or the expression cassette.

[0024] The present invention also provides a double-stranded ribonucleic acid molecule, wherein the double-stranded ribonucleic acid molecule comprises at least one dsRNA having an annealed complementary strand, the dsRNA having a sequence at least partially complementary to a target nucleotide sequence of a target gene of a coleopteran pest, the double-stranded ribonucleic acid molecule being produced by expression of the polynucleotide or the polynucleotide composition, wherein contact of the dsRNA with the coleopteran pest inhibits the expression of an endogenous nucleotide sequence specifically complementary to at least one strand of the dsRNA.

[0025] Furthermore, the double-stranded ribonucleotide molecule kills adult coleopteran pests after contact with the adult pests, or inhibits the growth, egg laying and / or hatching of offspring of the adult pests.

[0026] The present invention also provides a composition for controlling coleopteran pests, comprising one or more double-stranded ribonucleic acid molecules and at least one suitable carrier, excipient or diluent.

[0027] Furthermore, the two or more double-stranded RNA molecules may be present on the same nucleic acid construct, on different nucleic acid constructs, or any combination thereof.

[0028] Furthermore, at least a second insecticide is included for controlling coleopteran pests or other insects.

[0029] Alternatively, the second insecticide is selected from a Bacillus thuringiensis insecticidal protein, potato glycoprotein, a protease, an engineered antibody or antibody fragment, or a chitinase.

[0030] The present invention also provides a method for reducing the expression of a target gene in a coleopteran pest or inhibiting the growth of a coleopteran pest, wherein the coleopteran pest ingests or contacts the double-stranded RNA molecule, which acts to inhibit the biological function of the pest; preferably, the target gene is the polynucleotide or the polynucleotide composition.

[0031] The present invention also provides a method for controlling coleopteran pest infestation, which comprises contacting the coleopteran pest with an effective amount of at least one double-stranded RNA molecule or the composition.

[0032] The present invention also provides a method for increasing plant yield, which comprises introducing a construct comprising the polynucleotide, the polynucleotide composition, the expression cassette, the transformation vector, or the double-stranded ribonucleic acid sequence into a host plant of a coleopteran pest to produce a transgenic plant; and cultivating the plant to allow expression of at least one polynucleotide; wherein expression of the at least one polynucleotide kills coleopteran adults, or inhibits the growth, egg laying, and / or offspring hatching of the adults, as well as yield losses caused by coleopteran pest infestation.

[0033] Furthermore, the at least one polynucleotide is expressed to produce dsRNA, which inhibits target genes of at least a portion of pests that have contacted the host plant.

[0034] The present invention also provides a method for reducing the coleopteran pest population on a transgenic plant, the method comprising: the transgenic plant expresses a Cry insecticidal protein, a Vip insecticidal protein, a protease inhibitor, a lectin, an α-amylase or a peroxidase; and a construct comprising the polynucleotide or the polynucleotide composition or the expression cassette or the transformation vector or the double-stranded ribonucleic acid sequence is introduced into the transgenic plant to allow expression of at least one polynucleotide, wherein the at least one polynucleotide is expressed to produce dsRNA, and the dsRNA molecule inhibits the expression of a target gene in adult coleopteran pests, thereby killing the adult coleopterans, or inhibiting the growth, egg laying and / or offspring hatching of the adults, and reducing the number of coleopteran pest populations.

[0035] The present invention also provides a transgenic seed, which is a transgenic seed of the transgenic plant.

[0036] The present invention also provides a commercial product, which is derived from the transgenic plant or a part thereof.

[0037] Alternatively, the plant is soybean, wheat, barley, corn, tobacco, rice, rapeseed, cotton or sunflower.

[0038] Furthermore, the coleopteran pest is the two-spotted leaf beetle.

[0039] Detailed Description of the Invention

[0040] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present invention, the definitions and explanations of relevant terms are provided below.

[0041] The present invention comprises a method for regulating or inhibiting the expression of one or more target genes in a coleopteran pest, comprising: introducing part or all of a stabilized double-stranded RNA (e.g., dsRNA) or a modified form thereof (e.g., a small interfering RNA sequence) into cells in an invertebrate insect pest or into the extracellular environment. Within the insect, the dsRNA or siRNA enters the cells and inhibits the expression of at least one or more target genes, and this inhibition reduces the insect's ability to survive, grow, reproduce, and invade a host.

[0042] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0043] The terms "polynucleotide," "nucleotide," and "nucleic acid" are used interchangeably and include DNA, RNA, or hybrids thereof, which may be double-stranded or single-stranded. "Nucleic acid" may also contain non-naturally occurring or altered bases that allow for correct reading by a polymerase without reducing expression of a polypeptide encoded by the nucleic acid.

[0044] The terms "nucleotide sequence" and "nucleic acid sequence" both refer to the order of bases in DNA or RNA, and refer to the sense and antisense strands of a nucleic acid that exist as a single strand or in a duplex.

[0045] Those skilled in the art can readily mutate the DNA fragments of the present invention using known methods, such as directed evolution and point mutagenesis. Artificially modified fragments that share at least 90%, 95%, 98%, 98.5%, 99%, 99.5%, or 99.8% nucleotide identity with the aforementioned DNA fragments of the present invention and that possess the same function are derived from and are equivalent to the nucleotide sequences of the present invention.

[0046] The term "isolated", when referring to a nucleic acid, refers to a nucleic acid that is separated from a substantial portion of the genome in which it is naturally present and / or substantially separated from other cellular components that naturally accompany the nucleic acid. For example, any nucleic acid that has been produced by synthesis (e.g., by continuous base condensation) is considered to be isolated. Similarly, recombinantly expressed nucleic acids, cloned nucleic acids, nucleic acids produced by primer extension reactions (e.g., PCR), or nucleic acids that have otherwise been excised from a genome are also considered to be isolated.

[0047] A "reverse complementary sequence" refers to a sequence that is in the opposite direction but complementary to the original polynucleotide sequence.

[0048] The term "identity" refers to sequence similarity to a natural nucleic acid sequence. Identity can be assessed visually or using computer software. Using computer sequence alignment software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences.

[0049] As used herein, the term "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences may refer to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

[0050] Methods of alignment of sequences for comparison are well known to those of skill in the art and can be determined using the BLAST algorithm (Altschul et al., 1990, Mol. Biol. 215:403-10) available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ) using default parameters.

[0051] Various programs and alignment algorithms are described, for example, in Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearson et al. (1994) Methods Mol.Biol.24:307-31; Tatiana et al. (1999) FEMS Microbiol.Lett.174:247-50. Detailed considerations of sequence alignment methods and homology calculations can be found, for example, in Altschul et al. (1990) J. Mol.Biol.215:403-10.

[0052] The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available at the National Center for Biotechnology Information (Bethesda, MD) and on the Internet for use with several sequence analysis programs. A description of how to use this program to determine sequence identity is available under the "Help" section of BLAST™ on the Internet. To compare nucleic acid sequences, the "Blast 2 Sequences" function of the BLAST™ (Blastn) program can be used using default parameters. When evaluated in this way, nucleic acid sequences that have greater similarity to a reference sequence will show an increase in the percent identity.

[0053] It is also clear to those skilled in the art that the structure of a protein can be changed without adversely affecting its activity and functionality. For example, one or more conservative amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional configuration of the protein molecule. Examples and embodiments of conservative amino acid substitutions are clear to those skilled in the art. Specifically, an amino acid residue can be replaced with another amino acid residue belonging to the same group as the site to be replaced, i.e., a non-polar amino acid residue can be substituted for another non-polar amino acid residue, a polar uncharged amino acid residue can be substituted for another polar uncharged amino acid residue, a basic amino acid residue can be substituted for another basic amino acid residue, and an acidic amino acid residue can be substituted for another acidic amino acid residue. As long as the substitution does not impair the biological activity of the protein, conservative substitutions in which one amino acid is replaced by another amino acid belonging to the same group fall within the scope of the present invention. In addition, the present invention also encompasses mutant proteins that also contain one or more other non-conservative substitutions, as long as the non-conservative substitutions do not significantly affect the desired function and biological activity of the protein of the present invention.

[0054] As is well known in the art, one or more amino acid residues can be deleted from the N and / or C terminus of a protein while still retaining its functional activity. Therefore, on the other hand, the present invention also relates to fragments in which one or more amino acid residues are deleted from the N and / or C terminus of a mutant protein while retaining its desired functional activity, which are also within the scope of the present invention and are referred to as biologically active fragments. In the present invention, a "biologically active fragment" refers to a portion of a mutant protein of the present invention that retains the biological activity of the mutant protein of the present invention. For example, a biologically active fragment of a mutant protein can be a portion in which one or more (e.g., 1-50, 1-25, 1-10 or 1-5, such as 1, 2, 3, 4 or 5) amino acid residues are deleted from the N and / or C terminus of the protein, but which still retains the biological activity of the full-length protein.

[0055] The term "homology" refers to the level of similarity (sequence similarity or identity) between two nucleic acid or amino acid sequences in terms of nucleotide and amino acid identity or similarity. Homology, homologues and homology also refer to the concept of similar functional properties between different nucleic acids or proteins. Homologues include the genes of orthologues and paralogues. Homologues can be determined in one or more of the following ways by using the coding sequences disclosed herein or the homologues found in appropriate databases (e.g., in NCBI or other databases). For amino acid sequences, an algorithm should be used to compare sequences. For nucleotide sequences, the sequence of a DNA molecule can be compared with the sequence of a known or presumed homologue in almost the same manner. Homologs are at least 20% identical, or at least 30% identical, or at least 40% identical, or at least 50% identical, or at least 60% identical, or at least 70% identical, or at least 80% identical, or at least 88% identical, or at least 90% identical, or at least 92% identical, or at least 95% identical across any substantial region of the molecule (DNA, RNA, or protein molecule).

[0056] The term "complementary" or "complementarity" refers to the natural binding of polynucleotides by base pairing under permissive salt and temperature conditions. The complementarity between two single-stranded molecules can be "partial," where only some of the nucleotides bind, or it can be complete when there is complete complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands. "Complementary" polynucleotides are those that can base pair according to the standard Watson-Crick complementarity rules.

[0057] The term "substantially complementary" or "partially complementary" means that two nucleic acid sequences are complementary at at least about 50%, 60%, 70%, 80% or 90% of their nucleotides.

[0058] The term "hybridize" refers to two nucleotide sequences that hybridize to each other under stringent conditions; the two nucleotide sequences may also be considered to be substantially identical. In representative embodiments, two nucleotide sequences that are considered to be substantially identical hybridize to each other under high stringency conditions. If the proteins encoded by nucleic acids that do not hybridize to each other under stringent conditions are substantially identical, they are still substantially identical (e.g., due to the degeneracy of the genetic code).

[0059] As used herein, "expression cassette" refers to a nucleic acid molecule capable of directing the expression of at least one polynucleotide of interest (e.g., encoding a dsRNA molecule of the present invention) in an appropriate host cell, comprising a promoter operably linked to the polynucleotide of interest (which is operably linked to a termination signal). An "expression cassette" typically also comprises additional polynucleotides required for correct translation of the polynucleotide of interest. The expression cassette may also be included in other polynucleotides that are not required for direct expression of the polynucleotide of interest but exist as a convenient restriction site for removing the expression cassette from an expression vector.

[0060] The term "gene" is a defined region within a genome that, in addition to the aforementioned coding sequence, contains other key regulatory nucleic acid sequences responsible for the expression control (i.e., transcription and translation) of the coding portion. A gene may also contain additional 5' and 3' untranslated sequences and termination sequences. Other elements, such as introns, may also be present.

[0061] As used herein, the term "genome" refers to the entire complement of genetic material (genes and non-coding sequences) present in every cell or virus or organelle of an organism and / or the complete set of chromosomes inherited as a unit (haploid) from one parent.

[0062] The term "transgene" is used herein to describe genetic material that has been or will be artificially introduced into the genome of a host organism and is passed on to the host's offspring. A transgene will typically comprise a polynucleotide comprising non-coding and / or coding sequences that typically, but not necessarily, affect or cause an activity (e.g., regulation of transcription or translation, production of nucleotide sequences comprising coding and / or non-coding sequences, etc.).

[0063] In the present invention, the MhCOPI gamma and Mhmi2 genes can be introduced into plants according to methods commonly used in the industry, and can be subjected to transgenic manipulation via appropriate plant transformation expression vectors.

[0064] In some embodiments of the present invention, the dsRNA comprises a dsRNA containing a short hairpin RNA (shRNA) molecule. Expression of shRNA in cells is typically achieved by delivering a plasmid or recombinant vector into a transgenic plant (such as transgenic corn).

[0065] The term "dsRNA" or "RNAi" refers to a polyribonucleotide structure formed by a single self-complementary RNA chain or at least by two complementary RNA chains. The degree of complementarity (in other words, % identity) does not necessarily need to be 100%. Instead, it must be enough to allow the formation of a double-stranded structure under the conditions adopted. The term "completely complementary" refers to that all bases of the nucleotide sequence of the dsRNA are complementary or 'matching' with the bases of the target nucleotide sequence. The term "at least partially complementary" refers to that there is less than 100% matching between the bases of the dsRNA and the bases of the target nucleotide sequence. It will be understood by those skilled in the art that in order to mediate the downward regulation of target gene expression, the dsRNA only needs to be at least partially complementary to the target nucleotide sequence. As known in the art, RNA sequences with insertions, deletions and mispairings relative to the target sequence can still be effective in RNAi. According to the present invention, it is preferred that the dsRNA and the target nucleotide sequence of the target gene share at least 80% or 85% sequence identity, preferably at least 90% or 95% sequence identity, or more preferably at least 97% or 98% sequence identity, and even more preferably at least 99% sequence identity. Alternatively, the dsRNA may contain 1, 2, or 3 mismatches as compared to the target nucleotide sequence over each length of the 24 partially complementary nucleotides. One of ordinary skill in the art will appreciate that the degree of complementarity shared between the dsRNA and the target nucleotide sequence may vary depending on the target gene to be downregulated or on the insect pest species whose gene expression is to be controlled.

[0066] The target nucleotide sequence can be selected from any suitable region or nucleotide sequence of the target gene or its RNA transcript.For example, the target nucleotide sequence can be located within the 5'UTR or 3'UTR of the target gene or RNA transcript, or within the exon or intron region of the gene.

[0067] The dsRNA used in the present invention may comprise one or more dsRNAs, each of which comprises or consists of a nucleotide sequence that is at least partially complementary to a target nucleotide sequence in a target gene and, upon ingestion by an insect pest, acts to downregulate the expression of the target gene. The dsRNAs may be arranged as a single continuous region of the dsRNA or may be separated by the presence of a linker sequence. The linker sequence may comprise a short, random nucleotide sequence that is not complementary to any target nucleotide sequence or target gene.

[0068] As used herein, "Coleoptera" or "Coleoptera pest" refers to any member of the order Coleoptera, including coleopteran pests on plants. Non-limiting examples of coleopteran pests according to the present invention include species such as Diabrotica bispotata (Northern Corn Rootworm; NCR), Diabrotica occidentalis (Western Corn Rootworm; WCR), and Diabrotica elevenspotata (Southern Corn Rootworm; SCR).

[0069] The present invention describes the two-spotted leaf beetle, a member of the order Coleoptera, family Chrysomelidae, which is widely distributed in Northeast China, North China, Jiangsu, Zhejiang, Hubei, Jiangxi, Fujian, Guangdong, Guangxi, Ningxia, Gansu, Shaanxi, Sichuan, Yunnan, Guizhou, and Taiwan. It produces one generation annually, laying scattered eggs that overwinter beneath the topsoil. The larvae hatch in early to mid-May of the following year. They remain in the soil, feeding on the roots of grass crops or weeds. They pupate in the soil after 30-40 days, with a pupal period of 7-10 days. Newly emerged adults live on weeds along the edges of fields before migrating into cornfields. Populations begin to increase in early July, peaking in mid-to-late July, and the insects continue to cause damage until September. Adults are adept at flying and jumping, reaching distances of 3-5 meters or even greater. They prefer to fly and feed between 9:00 and 11:00 and 16:00 and 19:00. They are active on corn leaves and ears during the day, hiding on the undersides of leaves, in the heart leaves, and in soil crevices in the morning, evening, and midday.

[0070] The infestation habits of the two-spotted leaf beetle are characterized by adult insects being primarily active between July and August. They tend to congregate on the same plants or crops in the same area, particularly damaging young leaves and inflorescences. These adults feed on the mesophyll, leaving behind web-like or hole-like lesions. In crops such as corn and sorghum, they also feed directly on filaments and young grains, severely impacting crop yield and quality. The activity and infestation behavior of the two-spotted leaf beetle are affected by temperature, being most active above 15°C. In strong sunlight or high temperatures, the beetle hides on the underside of leaves or inflorescences to avoid direct exposure. Large-scale infestations by this pest can lead to widespread yield reductions or even complete crop failure. Integrated management measures, including physical, chemical, and biological control methods, are necessary. These include using insecticidal lamps to attract and kill adult insects, using chemical pesticides during critical growth periods, and employing biological control methods such as biotechnology to reduce the use of chemical pesticides and protect the environment and ecological balance.

[0071] As used herein, "controlling" insects means inhibiting the ability of one or more insect pests to survive, grow, feed, and / or reproduce, or limiting insect-related damage or crop plant loss, by silencing genes critical for pest survival, and then genes critical for reproduction. "Controlling" insects may or may not mean killing the insects, although it preferably does. Compositions for controlling target insects have insecticidal activity against the target insects.

[0072] The term "effective amount" refers to a concentration of dsRNA that triggers systemic nucleic acid silencing, resulting in the death or inability of target insects to reproduce.

[0073] The term "suitable carrier, excipient, or diluent" includes adjuvants, mixtures, enhancers, and the like that facilitate the administration of an active ingredient (e.g., a dsRNA molecule of the invention). Suitable carriers should not be phytotoxic to valuable crops, particularly at the concentrations used when the composition is applied in the presence of crops, and should not chemically react with the active ingredient compounds herein (i.e., the dsRNA of the invention or other composition components). Such mixtures can be designed for direct application to crops, or can be concentrates or formulations that are typically diluted with additional carriers and adjuvants prior to application.

[0074] The composition of the present invention is formulated to include at least one other agronomic agent, such as a herbicide or a other insecticide. As used herein, 'second insecticide' or 'other insecticide' refers to an insecticide other than the first or dsRNA molecule of the composition. Alternatively, the composition of the present invention can be delivered in combination with at least one other agronomic agent (such as a herbicide or a second insecticide). The other insecticide can be selected from any insecticide known in the art and / or can include an interfering RNA that acts to lower the expression of a target gene in the pest species after being ingested by a pest. For example, dsRNA and the second insecticide can target different insect pest species or can target pest organisms of different families or classes, such as fungi or nematodes or insects. One of ordinary skill in the art will appreciate how to test the combination of dsRNA molecules with other agronomic agents with respect to synergistic effects. The composition comprises a first dsRNA molecule described elsewhere herein and one or more additional pesticides, each toxic to the same insect pest, wherein the one or more additional pesticides are optionally and without limitation a Bacillus thuringiensis insecticidal protein, a potato glycoprotein, a protease, an engineered antibody or antibody fragment, or a chitinase.

[0075] The present invention encompasses a nucleic acid construct or vector comprising a dsRNA of the present invention. The present invention further encompasses nucleic acid molecules encoding at least one dsRNA molecule of the present invention. The present invention further encompasses nucleic acid constructs comprising at least one dsRNA molecule of the present invention, or comprising nucleic acid molecules encoding at least one dsRNA molecule of the present invention. The present invention further encompasses nucleic acid constructs wherein the nucleic acid construct is an expression vector. The present invention further encompasses recombinant vectors comprising regulatory sequences operably linked to the nucleotide sequence encoding the dsRNA molecule of the present invention. Regulatory sequences may refer to promoters, enhancers, transcription factor binding sites, insulators, silencers, or any other DNA elements involved in gene expression.

[0076] The term "transfer" is a method for introducing a heterologous nucleic acid into a host cell or organism. Specifically, "transfer" refers to the stable integration of a DNA molecule into the genome of the organism of interest.

[0077] The term "transferred / transgenic / recombinant" refers to a host organism such as a bacterium or plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the host's genome, or the nucleic acid molecule can also exist as an extrachromosomal molecule. Such extrachromosomal molecules can replicate autonomously. The transformed cells, tissues or plants should be understood to encompass not only the end product of the transformation process, but also their transgenic progeny. A "non-transferred", "non-transgenic" or "non-recombinant" host refers to a wild-type organism, such as a bacterium or plant, that does not contain the heterologous nucleic acid molecule.

[0078] In transgenic plants, the expression of the dsRNA molecule is driven by the regulatory sequence of the promoter that is included in the plant. The selection of the promoter will depend on the time and space required for expression and vary, and also depends on the insect target species and varies. Therefore, the expression of the dsRNA of the present invention in leaves, stalks or stems, spikes, inflorescences (such as spikes, panicles, cobs, etc.), roots and / or seedlings has been considered. In many cases, researchers hope to protect more than one type of insect pests and express them in multiple tissues. Although it has been shown that many promoters from dicots are operable in monocots and vice versa, it is ideal to select dicot promoters for expression in dicots and select monocot promoters for expression in monocots. However, there is no restriction on the origin of the selected promoter; as long as they are feasible in driving the expression of dsRNA or siRNA in target cells.

[0079] Promoters suitable for use in the present invention include, but are not limited to, those that constitutively drive the expression of a nucleotide sequence, those that drive expression when induced, and those that drive expression in a tissue or development-specific manner. These different types of promoters are known in the art.

[0080] The present invention can also optionally be included in a transcription and / or translation termination region (i.e., termination region) with functionality in plants. A variety of transcription terminators are available for use in expression cassettes and are responsible for transcription termination and correct mRNA polyadenylation when exceeding interested heterologous nucleotide sequence. This termination region can be natural for this transcription initiation region, can be natural for this operably connected nucleotide sequence of interest, can be natural for this plant host, or can be derived from another source (i.e., external or heterologous for this promotor, this nucleotide sequence of interest, this plant host, or its arbitrary combination). Suitable transcription terminators include but are not limited to CAMV 35S terminator, tml terminator, nopaline synthase terminator, and / or pea rbcs E9 terminator. These terminators can be used in both monocotyledons and dicotyledons. In addition, the natural transcription terminator of the encoding sequence can be used.

[0081] The present invention can also include polynucleotides encoding other desired proterties. Such desired proterties can be other polynucleotides that confer insect resistance or virus resistance, or other desired proterties in agriculture. Such polynucleotides can be stacked with any combination of nucleotide sequences to produce plants, plant parts or plant cells with desired phenotypes, such as Cry insecticidal proteins, Vip insecticidal proteins, protease inhibitors, lectins, α-amylases or peroxidases. The stacked combination can be produced by any method, including but not limited to, by any conventional cross-breeding plants or by genetic transformation. If stacking is performed by genetic transformation of these plants, the nucleotide sequences encoding other desired proterties can be combined at any time and in any order. For example, a single transgenic plant can include multiple expression cassettes so that multiple expression cassettes are introduced into the genome of the transformed cell at a single genomic location, i.e., molecular stacking technology. Alternatively, a transgenic plant comprising one or more desired proterties can be used as a target for introducing other proterties by subsequent transformation. Additional nucleotide sequences can be introduced simultaneously in a co-transformation protocol with the nucleotide sequences, nucleic acid molecules, nucleic acid constructs, and / or other compositions of the present invention provided by any combination of expression cassettes.

[0082] The agrobacterium-mediated transformation that the present invention uses, binary vector or the carrier that carries at least one T-DNA border sequence are applicable to, and for direct gene transfer, any carrier all is applicable to, and the linear DNA that only contains interested construct is perhaps preferred.In the situation that direct gene transfer, can use conversion or cotransformation with single DNA kind.For direct gene transfer and agrobacterium-mediated transformation these two, conversion is usually (but not necessarily) carried out with a kind of selected marker, and this selected marker can provide the resistance for a kind of antibiotic (kanamycin, hygromycin or methotrexate) or a kind of weed killer herbicide (Basta).Plant transformation vector of the present invention can also comprise other selected marker genes, comprise the gene (as phosphomannose isomerase (pmi)) (being incorporated into this by reference) that transgenic plant positive selection is provided or the phosphinothricin ammonium acetyltransferase (pat) to weed killer herbicide phosphinothricin ammonium (phosphinothricin) tolerance is provided.But the selection of selected marker is not crucial for the present invention.

[0083] The transgenic plant expressing the dsRNA of the present invention has tolerance or resistance to being attacked by target insect pests. When the insect starts to ingest such a transgenic plant, it also ingests the expressed dsRNA. The ingested dsRNA is processed by its so-called Dicer nuclease in the insect body and becomes a lot of 21-25bp siRNA. The siRNA targets and destroys the target gene mRNA, causing the expression level of the key gene to be significantly reduced, eventually leading to the death of the insect, or unable to reproduce normally. This can hinder the insect from further biting plant tissue or even injuring or killing the insect. The nucleotide sequence encoding the dsRNA of the present invention is inserted into a kind of expression cassette, and then the expression cassette is preferably stably integrated into the genome of the plant. These nucleotide sequences of the expression cassette introduced into the plant genome are heterologous to the plant and are naturally occurring. Plants transformed according to the present invention can be monocots or dicots and include, but are not limited to, corn, wheat, barley, rye, sweet potatoes, beans, peas, chicory, lettuce, cabbage, cauliflower, broccoli, turnips, radishes, spinach, asparagus, onions, garlic, peppers, celery, pumpkins, squashes, hemp, zucchini, apples, pears, quinces, melons, plums, cherries, peaches, nectarines, apricots, strawberries, grapes, raspberries, blackberries, pineapples, avocados, papayas, mangoes, bananas, soybeans, tomatoes, millet, sorghum, sugarcane, sugar beets, sunflowers, rapeseed, clover, tobacco, carrots, cotton, alfalfa, rice, potatoes, eggplants, cucumbers, Arabidopsis, and woody plants such as conifers and deciduous trees. In further embodiments, the transgenic plant is a transgenic corn plant.

[0084] The present invention encompasses biological samples from transgenic plants of the present invention, seeds, or parts thereof, wherein the part comprises a nucleic acid as at least one chain of a dsRNA of the present invention or encoding a dsRNA of the present invention. In other embodiments, the present invention encompasses a commodity product derived from a transgenic plant of the present invention, seed, or parts thereof. In certain embodiments, the commodity product is selected from the group consisting of whole or treated seeds, beans, cereals, grains, shells, powders, coarsely milled hulled cereals, flour, sugar, sugar, starch, protein concentrates, protein isolates, waxes, oils, extracts, juices, concentrates, liquids, syrups, feed, silage, fiber, paper, or other food or products produced from plants. In other embodiments, the biological sample or commodity product are toxic to insects. In other embodiments, the transgenic plant is a transgenic corn plant.

[0085] The isolated polynucleotide provided by the present invention has the following advantages:

[0086] 1. Internal control. Existing technologies primarily rely on physical, chemical, or biological methods. However, the present invention controls the beetle by producing dsRNA in the plant body that kills it, effectively controlling it through internal factors.

[0087] 2. Combined control of two generations. The direct lethal gene (MhCOPI gamma) and the reproductive control gene (Mhmi2) are used together to "eliminate" pests. Modern leaf beetles, such as the two-spotted leaf beetle, are killed when feeding on transgenic crops expressing dsRNA. Even if they survive, the surviving pests will produce significantly fewer eggs or their eggs will not hatch, directly affecting the survival of the next generation.

[0088] 3. Environmentally friendly. Currently commonly used chemical control methods can cause environmental pollution. Using dsRNA for control can reduce the development of pesticide resistance and pesticide residue problems.

[0089] 4. High specificity: The polynucleotides used to control coleopteran pests of the present invention do not affect the expression of non-target sequences in the host. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1. Schematic diagram of the MhCOPI gamma gene, Mhmi2 gene, and dsRNA targets.

[0091] Figure 2. Schematic diagram of the mGFP5 gene and dsRNA target.

[0092] Figure 3. Survival rate over time of adults of the two-spotted leaf beetle fed with MhCOPI gamma dsRNA and Mhmi2 dsRNA alone or in combination.

[0093] Figure 4. Egg production of female adults of the two-spotted leaf beetle fed with MhCOPI gamma dsRNA and Mhmi2 dsRNA alone or in combination.

[0094] Figure 5. Hatching rate of female adults of the two-spotted leaf beetle fed with MhCOPI gamma dsRNA and Mhmi2 dsRNA alone or in combination.

[0095] Figure 6. Relative expression levels of endogenous genes after feeding MhCOPI gamma dsRNA to adults of the two-spotted leaf beetle for 3 days.

[0096] Figure 7. Relative expression levels of endogenous genes after feeding Mhmi2 dsRNA to adults of the two-spotted leaf beetle for 3 days.

[0097] Figure 8. Map of transgenic maize vector pQY010775.

[0098] Figure 9. Mortality of adult leaf beetles 10 days after feeding on transgenic maize materials expressing MhCOPI gamma dsRNA+Mhmi2 dsRNA, positive events Event-1, 2, 3, 4, 5, 6, transgenic negative materials N-1, N-2, N-3, and non-transgenic recipient maize materials.

[0099] Figure 10. Endogenous target gene expression levels in adults of the two-spotted leaf beetle feeding on resistant plants expressing MhCOPI gamma dsRNA + Mhmi2 dsRNA transgenic event Event-3 three days after ingestion.

[0100] Sequence Description DETAILED DESCRIPTION

[0101] The present invention is further described below with reference to examples. The following description is by way of examples, but the scope of protection of the present invention should not be limited thereto.

[0102] Example 1: Identification of RNAi target genes in the two-spotted leaf beetle

[0103] By analyzing the transcriptome of the two-spotted firefly beetle (Lepidoptera: Le ...

[0104] The MhCOPI gamma and Mhmi2 sequences were analyzed to identify targeting dsRNA sequences. A full-sequence scanning strategy was employed to identify the optimal target for transgenic transgenesis. dsRNAs 1-19 (SEQ ID NO: 3-SEQ ID NO: 21) targeting MhCOPI gamma were obtained by PCR amplification. A combination of Mhmi2 dsRNA ts1 (SEQ ID NO: 24) and Mhmi2 dsRNA ts2 (SEQ ID NO: 25) targeting Mhmi2 was selected, with the corresponding DNA sequence being SEQ ID NO: 23. A schematic diagram of the gene and its target is shown in Figure 1. mGFP5 (SEQ ID NO: 28) and its dsRNA (the corresponding DNA sequence is shown in SEQ ID NO: 29) were used as controls. A schematic diagram of the gene and its target is shown in Figure 2.

[0105] Example 2: Artificial Synthesis of dsRNA

[0106] The full-length MhCOPI gamma was amplified using the cDNA of the two-spotted leaf beetle as a template using primers of SEQ ID NO: 38 and SEQ ID NO: 39. The amplified product was purified by agarose gel extraction kit (Tiangen, DP209-03) and ligated to the vector using the pClone007Versatile Simple Vector Kit (TSINGKE, TSV-007VS). 5α competent cells (TSINGKE, TSC-C01) were plated onto ampicillin (Amp)-resistant LB solid screening medium. Positive colonies were picked and sequenced in ampicillin-resistant liquid LB medium. The full-length sequence of the target gene MhCOPI gamma was obtained, and the nucleotide sequence is shown in SEQ ID NO: 1.

[0107] Using the sequenced plasmid as a template, primers (SEQ ID NO: 40 to SEQ ID NO: 77) containing the T7 promoter sequence were used to amplify 19 target sequences of MhCOPI gamma. The purified amplified products were recovered and used as templates for dsRNA synthesis. dsRNA was synthesized according to the instructions of the T7 RNAi Transcription Kit (Vazyme, TR102-01). Synthesis quality and band size were verified by 2% agarose gel electrophoresis to obtain MhCOPI gamma dsRNAs 1-19 (sequence length range 88-169 bp). MhCOPI gamma dsRNA (ts1+ts2) (sequence length 325 bp) and Mhmi2 dsRNA (ts1+ts2) (sequence length 345 bp) were synthesized using the same method. The concentrations of these dsRNAs were determined using a NanoDrop 2000 (Thermo Scientific). The dsRNAs were aliquoted and stored at -80°C until use.

[0108] Example 3 Effects of MhCOPI gamma dsRNA and Mhmi2 dsRNA, alone or in combination, on the survival rate, egg production, and egg hatching rate of the two-spotted leaf beetle

[0109] Bioassays for the two-spotted leaf beetle were performed using artificial diets mixed with each dsRNA. Pure water and mGFP5 dsRNA were used as controls. MhCOPI gamma dsRNA, Mhmi2 dsRNA, MhCOPI gamma dsRNA and Mhmi2 dsRNA were mixed with pure water, and mGFP5 dsRNA was prepared at 218.75 ng / mL for later use. Eight tube caps were prepared and placed in a clean bench. The artificial diet was filled into each of the eight tube caps (the surface area of ​​each tube cap was approximately 0.125 cm). 2 10 μL of the above-prepared solution was added to the feed surface of each eight-tube strip cap, with a final treatment concentration of 17.5 ng / cm 2 Unmated female and male individuals of the two-spotted firefly leaf beetle were collected, and 20 pairs of adults were placed in a bioassay device for mating. After mating, the two-spotted firefly leaf beetles began to be fed the above-mentioned feed containing dsRNA. The feed containing the same concentration of dsRNA was replaced every other day, and the survival of the adults was counted every day. On the 16th day, the surviving female adults were transferred to the egg-laying device, and the feed without dsRNA was replaced. Eggs were laid for 8 days, and the female adults were removed on the 24th day. The number of eggs laid was counted and stored on moisturizing filter paper. Starting from the 40th day, the number of larvae hatched was counted every day. The cumulative survey was conducted until the 60th day, and the hatching rate was calculated. Each treatment was repeated 3 times.

[0110] Compared with the control, the above-mentioned MhCOPI gamma dsRNA, Mhmi2 dsRNA, and the mixture of MhCOPI gamma dsRNA and Mhmi2 dsRNA had significant effects on the survival rate, egg production and / or egg hatching rate of two-spotted firefly beetles. Representative results are shown in Figures 3-5. Compared with the control group mGFP5 dsRNA and pure water treatment, the survival rate of two-spotted firefly beetles fed MhCOPI gamma dsRNA (ts1+ts2) and MhCOPI gamma dsRNA (ts1+ts2) mixed with Mhmi2 dsRNA (ts1+ts2) was significantly reduced (Figure 3). Compared with the control group of mGFP5 dsRNA and pure water treatment, the egg production (Figure 4) and hatching rate (Figure 5) of adults of the two-spotted firefly leaf beetle fed MhCOPI gamma dsRNA (ts1+ts2), Mhmi2 dsRNA (ts1+ts2), and a mixture of MhCOPI gamma dsRNA (ts1+ts2) and Mhmi2 dsRNA (ts1+ts2) decreased to varying degrees. Compared to feeding MhCOPI gamma dsRNA (ts1+ts2) alone, feeding Mhmi2 dsRNA (ts1+ts2) alone had a greater impact on egg production and hatching rate of adult two-spotted leaf beetles. The most significant effect was achieved when MhCOPI gamma dsRNA (ts1+ts2) and Mhmi2 dsRNA were mixed, with both egg production and hatching rates falling below 5%. These results suggest that feeding MhCOPI gamma dsRNA and Mhmi2 dsRNA together can sustainably control the offspring of two-spotted leaf beetles.

[0111] Example 4: Analysis of the Inhibition of Target Gene Expression in MhCOPI gamma dsRNA and Mhmi2 dsRNA in the Two-spotted Firefly Leaf Beetle

[0112] Real-time fluorescence quantitative analysis was used to analyze the expression of target genes after feeding MhCOPI gamma dsRNA (ts1 + ts2) and Mhmi2 dsRNA (ts1 + ts2). Fluorescent quantitative primers for COPI gamma, mi2, and the internal reference gene RPS9 were synthesized, and the primer sequences are shown in Table 1.

[0113] Table 1 Fluorescence quantitative detection primers

[0114] 17.5 ng / cm was used for collection 2After 3 days of dsRNA feeding, RNA of the surviving two-spotted firefly leaf beetles was extracted by Trizol method. 1 μg of RNA of the above insects was reverse transcribed using Takara kit to obtain cDNA as template for fluorescence quantitative PCR, and RT-qPCR analysis was performed with the above primers.

[0115] The expression levels of COPI gamma and mi2 in each group of two-spotted firefly leaf beetles were counted after feeding MhCOPI gamma dsRNA (ts1+ts2) and Mhmi2 dsRNA (ts1+ts2) for 3 days.

[0116] Figures 6 and 7 show the results of quantitative PCR analysis using the primers listed in Table 1. Primers were positioned to avoid the MhCOPI gamma dsRNA (ts1+ts2) and Mhmi2 dsRNA (ts1+ts2) sequences to detect endogenous gene expression in the two-spotted firefly leaf beetle. Compared to the mGFP5 dsRNA and pure water controls, COPI gamma expression in the two-spotted firefly leaf beetle was reduced by 50-70% (3 days). Compared to the mGFP5 dsRNA and pure water controls, mi2 expression in the two-spotted firefly leaf beetle was reduced by 30-40% (3 days). The results showed that feeding MhCOPI gamma dsRNA and / or Mhmi2 dsRNA could cause RNAi effects on the corresponding genes in the two-spotted firefly leaf beetle, resulting in a significant decrease in the expression of the COPI gamma gene, a key gene responsible for vesicle transport from the endoplasmic reticulum to the Golgi apparatus, and a significant decrease in the expression of the mi2 gene encoding the CHD complex ATPase, thereby leading to the death or growth inhibition of the two-spotted firefly leaf beetle, and inhibiting the egg laying and hatching rate of the offspring.

[0117] Example 5. Construction of a vector expressing the combination of MhCOPI gamma dsRNA and Mhmi2 dsRNA in corn, genetic transformation, and screening of transgenic corn events

[0118] 1. Construction of corn dsRNA expression vector

[0119] Based on the Agrobacterium binary expression vector pCAMBIA1300, the vector was linearized with XhoI and the glufosinate selection marker gene BlpR was inserted through seamless cloning to generate the intermediate vector pCAMBIA1300-BlpR. The intermediate vector pCAMBIA1300-BlpR was linearized with HindIII and the ZmUbi1 promoter (AvrII+MluI) and PinII terminator were inserted through seamless cloning to create the maize dsRNA expression backbone vector.

[0120] The dsRNA backbone vector obtained above was linearized by double digestion with restriction endonucleases AvrII and MluI (purchased from NEB). Construction of the dsRNA expression vector required sequentially ligating the DNA sequence (SEQ ID NO: 26) fragment corresponding to MhCOPI gamma dsRNA (ts1 + ts2) + Mhmi2 dsRNA (ts1 + ts2), the ST-LS1L sequence (SEQ ID NO: 27), and the reverse complementary sequence fragment of the DNA sequence corresponding to the dsRNA into the vector by seamless cloning to obtain the final transformation vector pQY010775 of MhCOPI gamma dsRNA (ts1 + ts2) + Mhmi2 dsRNA (ts1 + ts2) (see Figure 8).

[0121] 2. Acquisition and molecular identification of genetically modified corn

[0122] The transformation vector pQY010775 was transferred into maize recipients via Agrobacterium-mediated immature embryo transformation to obtain transgenic maize plants. Leaves from the T0 generation transformed seedlings were used to extract genomic DNA from the plants using the CTAB method. Transgenic positive transformants were screened by molecular identification using PCR. The identification primers were designed for the loop region ST-LS1 that forms the RNA stem-loop structure. The specific sequences are shown in Table 2.

[0123] Table 2 PCR identification primers for T0 generation transgenic plants

[0124] Example 6: Insecticidal Effect of Transgenic Corn on the Two-spotted Leaf Beetle and Analysis of Target Gene Expression

[0125] Through screening, six transgenic-positive events (Event-1, 2, 3, 4, 5, and 6) were identified and self-pollinated to produce T1 seeds. These six transgenic-positive events, along with three molecularly negative events (N-1, 2, and 3), and wild-type recipient maize were selected for sowing to test their effects on the two-spotted leaf beetle. Ten plants per event were grown in the greenhouse to the V4 stage. Fifteen adult two-spotted leaf beetles were then seeded on leaves. For the first three days, they fed on the transformed material, followed by non-transgenic maize silk. Four replicates were set up for each event. On the tenth day, the number of surviving adults in each device was measured. As shown in Figure 9, the mortality rate of Event-3 was significantly higher than that of the other transgenic-positive events and the control group. Resamples were then collected for target gene verification. After three days of feeding on Event-3 leaves, the test insects were tested for knockdown of the endogenous target genes. RT-qPCR results showed that the expression of the endogenous target genes MhCOPI gamma and Mhmi2 was significantly reduced in two-spotted leaf beetles fed Event-3 transgenic material (Figure 10). The results showed that MhCOPI gamma dsRNA and Mhmi2 dsRNA were expressed in Event-3 transgenic corn event and could effectively inhibit the expression of target genes in the two-spotted firefly leaf beetle.

[0126] Example 7: Field evaluation of resistance of transgenic corn to the two-spotted leaf beetle

[0127] In July 2023, a plot where the two-spotted leaf beetle frequently occurs was selected at the Jilin transgenic base. A resistance identification pool was set up in the field. Event-3 (a transformant that showed resistance after indoor identification) was sown with 40 T1 plants in the experimental field as a plot. At the same time, 40 adjacent wild-type corn plants were used as control plots. The plots were surrounded by plastic film and breathable gauze on all sides. When the transgenic corn plants reached the V4-V5 leaf age, newly emerged adults were collected from the field, and 800 adults were inoculated into each plot. In May of the following year, wild-type control corn was sown in the two plots, and gauze was rebuilt to collect adults that emerged from the soil. In August, adults in the gauze were collected and recorded every 3 days, and the number of emerging adults of the next generation was counted.

[0128] Results showed that the cumulative number of emerged adults in the pQY010775 transgenic corn plots was zero, meaning no adults were collected; whereas a total of 772 adults were collected in the wild-type corn plots. These results indicate that the combination of MhCOPI gamma dsRNA and Mhmi2 dsRNA imparts sustained resistance to the two-spotted leaf beetle population in the corn plants, completely eliminating the beetles in the plots and demonstrating promising industrial value.

[0129] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was individually and specifically indicated to be incorporated by reference.

[0130] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims and are intended to be within the scope of the invention.

Claims

1. An isolated polynucleotide comprising at least one polynucleotide sequence selected from the group consisting of: (a) the polynucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 22; (b) a polynucleotide sequence having at least 87%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to at least 88, at least 156, at least 169, or at least 325 consecutive polynucleotides of SEQ ID NO: 1 or a polynucleotide sequence having at least 83% sequence identity to at least 144, at least 201, or at least 345 consecutive polynucleotides of SEQ ID NO: 22; (c) any one of the polynucleotide sequences shown in SEQ ID NO: 2 to SEQ ID NO: 21 or any one of the polynucleotide sequences shown in SEQ ID NO: 23 to SEQ ID NO: 26; (d) a polynucleotide sequence that hybridizes or is complementary to the polynucleotide sequence defined in any one of (a) to (c) above under stringent conditions.

2. A polynucleotide composition comprising any one of the following polynucleotide sequences or their complementary sequences: (a) the polynucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 22; (b) a polynucleotide sequence having at least 87%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to at least 88, at least 156, at least 169, or at least 325 contiguous polynucleotides of SEQ ID NO: 1 and at least 83% sequence identity to at least 144, at least 201, or at least 345 contiguous polynucleotides of SEQ ID NO: 22; (c) any one of the polynucleotide sequences shown in SEQ ID NO: 2 to SEQ ID NO: 21 and any one of the polynucleotide sequences shown in SEQ ID NO: 23 to SEQ ID NO: 25; Preferably, the composition comprises the polynucleotides shown in SEQ ID NO: 2 and SEQ ID NO: 23 or their complementary sequences; More preferably, the composition is the polynucleotide shown in SEQ ID NO: 26 or its complementary sequence.

3. A polynucleotide encoding a double-stranded ribonucleic acid molecule, comprising: (1) any polynucleotide sequence according to claim 1 or any polynucleotide composition sequence according to claim 2; (2) spacer sequences; and (3) The reverse complementary sequence of the polynucleotide sequence or polynucleotide composition sequence described in item (1); Preferably, the spacer sequence is SEQ ID NO:

27.

4. An expression cassette comprising the polynucleotide of claim 1 or 3 or the polynucleotide composition of claim 2 under the control of an operably linked regulatory sequence. 5 . A plant transformation vector comprising the polynucleotide according to claim 1 or 3 , the polynucleotide composition according to claim 2 , or the expression cassette according to claim 4 .

6. A double-stranded RNA molecule, wherein the double-stranded RNA molecule comprises at least one dsRNA having an annealed complementary strand, the dsRNA having a sequence at least partially complementary to a target nucleotide sequence of a target gene of a coleopteran pest, the double-stranded RNA molecule being produced by expression of the polynucleotide of claim 1 or 3 or the polynucleotide composition of claim 2, wherein contact of the dsRNA with the coleopteran pest inhibits expression of an endogenous nucleotide sequence specifically complementary to at least one strand of the dsRNA.

7. The double-stranded ribonucleic acid molecule according to claim 6, wherein the double-stranded ribonucleic acid molecule kills adult coleopteran pests after contact with the adult pests, or inhibits the growth, egg laying and / or offspring hatching of the adult pests.

8. A composition for controlling coleopteran pests, comprising one or two or more double-stranded ribonucleic acid molecules according to claim 6 or 7 and at least one suitable carrier, excipient or diluent.

9. The composition of claim 8, wherein the two or more double-stranded ribonucleic acid molecules can be present on the same nucleic acid construct, on different nucleic acid constructs, or any combination thereof.

10. A composition according to claim 8 or 9 further comprising at least a second insecticide for controlling coleopteran pests or other insects.

11. The composition of claim 10, wherein the second insecticide is selected from the group consisting of a Bacillus thuringiensis insecticidal protein, potato glycoprotein, a protease, an engineered antibody or antibody fragment, or a chitinase.

12. A method for reducing target gene expression in a coleopteran pest or inhibiting the growth of a coleopteran pest, wherein the coleopteran pest ingests or comes into contact with the double-stranded RNA molecule according to claim 6 or 7, which acts to inhibit the biological function of the pest; preferably, the target gene is the polynucleotide according to claim 1 or the polynucleotide composition according to claim 2.

13. A method for controlling coleopteran pest infestation, comprising contacting the coleopteran pest with an effective amount of at least one double-stranded RNA molecule according to claim 6 or 7 or a composition according to any one of claims 8 to 11.

14. A method for increasing plant yield, comprising: introducing a construct comprising the polynucleotide of claim 1 or 3, the polynucleotide composition of claim 2, the expression cassette of claim 4, the transformation vector of claim 5, or the double-stranded ribonucleic acid sequence of claim 6 or 7 into a host plant of a coleopteran pest to produce a transgenic plant; and cultivating the plant to allow expression of at least one polynucleotide; wherein expression of the at least one polynucleotide kills coleopteran adults, or inhibits growth, egg laying, and / or offspring hatching of the adults, as well as yield loss due to coleopteran pest infestation.

15. The method of claim 14, wherein expression of the at least one polynucleotide produces a dsRNA that inhibits a target gene in at least a portion of the pest that has contacted the host plant.

16. A method for reducing the population of coleopteran pests on transgenic plants, the method comprising: the transgenic plants expressing Cry insecticidal proteins, Vip insecticidal proteins, protease inhibitors, lectins, α-amylases or peroxidases; and introducing into the transgenic plants a construct comprising the polynucleotide of claim 1 or 3, the polynucleotide composition of claim 2, the expression cassette of claim 4, the transformation vector of claim 5, or the double-stranded ribonucleic acid sequence of claim 6 or 7, to allow expression of at least one polynucleotide, wherein the at least one polynucleotide is expressed to produce dsRNA, wherein the dsRNA molecules inhibit the expression of target genes in adult coleopteran pests, thereby killing the adult coleopterans, or inhibiting the growth, egg laying and / or offspring hatching of the adults, and reducing the population of the coleopteran pests.

17. A transgenic seed, which is a transgenic seed of the transgenic plant according to any one of claims 14 to 16.

18. A commodity product derived from the transgenic plant or part thereof according to any one of claims 14 to 17.

19. The method according to any one of claims 14 to 16, the seed according to claim 17, or the product according to claim 18, wherein the plant is soybean, wheat, barley, corn, tobacco, rice, rapeseed, cotton, or sunflower.

20. The double-stranded RNA molecule according to claim 6 or 7, the composition according to any one of claims 8 to 11, or the method according to any one of claims 12 to 16, wherein the coleopteran pest is Diabrotica bipunctata.

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