AU2016228052B2 - Uses of insecticidal protein - Google Patents

Abstract

Uses of insecticidal protein and a method for controlling chilo suppressalis insects. The method comprises: allowing chilo suppressalis insects to be at least in contact with a Vip3A protein. The chilo suppressalis insects are controlled by means of the Vip3A protein that is generated in plant bodies and that can kill chilo suppressalis insects. Compared with the agricultural control method, the chemical control method and the physical control method used in the prior art, the whole plants are protected at the total growth period so as to control invasion of the chilo suppressalis insects, and the insecticidal protein is pollution-free, residue-free, stable and thorough in effect, simple, convenient and economical.

Description

USES OF INSECTICIDAL PROTEIN

Cross-reference to related applications

The present application claims the priority of Chinese patent application No. 201510097004.0 filed on March 04, 2015, the full text of which is incorporated here by reference.

Field of the invention

The present invention relates to uses of insecticidal protein, in particular, involved in a Vip3A protein used in protecting plant form the harm of pest Chilo suppressalis by expressing the Vip3A protein in the plant.

Background of the invention

Chilo suppressalis belongs to Lepidoptera, Pyralidae, is one of the most common pests in the north and south rice regions of China. The incests can cause dry sheath, withered seedlings during tillering period, and may cause injury and white spike during ear stage, in general years may lead to 3%-5% reduction of yield, even more than 3 percent reduction of yield in severe case, thus cause a serious threat to current rice production.

Rice is important food crop in China, with the strengthening of the global greenhouse effect, the temperature has risen in the past two years, and the species and quantity of pests have been improved. Chilo suppressalis cause tremendous grain loss every years, it even affects the living conditions of the local populations. At present, agricultural control, chemical control and physical control are usually applied to control Chilo suppressalis.

Agricultural control is a method to comprehensively manage multiple factors of the whole farmland ecological system. By means of the regulation of crops, pests and the environmental factors, a farmland ecological environment is created, which is conducive to the crop growth and nonadvantagous to the outbreaking of Chilo suppressalis. Adjusting the rice sowing period, dealing with rice stubble, irrigating pests, pulling out the white spike and other measures can be applied to achieve the purpose of eliminating a certain number of pests. Because agricultural control must be subject to crop layout and production requirements, the application of agriculture

941 701 5_1 (GHMatters) P1 06623.AU control is limited and cannot serve as an emergency measures. It does not work when Chilo suppressalis outbreaks.

Chemical control, i.e. pesticides control, using chemical pesticides to kill pests, chemical control is an important part of the comprehensive treatment of Chilo suppressalis. It is rapid, convenient, simple and economically, chemical control is an indispensable measure for emergency when Chilo suppressalis outbreaks. Chilo suppressalis is a stem borer pest, it is very important to grasp the control period, the best period of applying pesticides is from the egg hatching stage and before the larvae drilling the stem, otherwise after the older larvae get into the stem, it will be very difficult to achieve the purpose of pest control. The current chemical control method is mainly liquid spray. But chemical control also has its limitations. For example, the improper operation can usually cause crop phytotoxicity, and pest resistance to drugs, in addition, natural enemies can also be reduced, chemical pesticides may cause the environmental pollution and destruct the farmland ecosystem as well, furthermore, pesticide residues may pose a threat to the safety of people and animals and leads to other serious results.

Physical control is to control pests by using physical factors, such as: light, electricity, color, humidity, temperature and etc as well as mechanical equipment to trap and kill the pests and sterilize the pests by irradiation based on the response of pests to physical factors in the environmental conditions. Currently, the most widely used method is trapping by using frequency vibration insecticidal lamp, the lamp utilizes the photo taxis of the insects, uses light at close distance, uses wave at long distance to lure pests getting close, it has a certain control effect on Chilo suppressalis, but the dirt on the the high-voltage grid of frequency vibration insecticidal lamp need to be cleaned up everyday, otherwise may affect the control effect, and the lamp cannot be open in thunderstorm days, and there are still under risk of hurting people by electric shork in operation, in addition, the one-time cost of instrallation of the lamp is large.

In order to solve the limitations of the agricultural control, chemical control and physical control in practical application, the scientists found that, by means of transfecting genes encoding pesticidal protein which is from Bacillus thuringiensis into plants, some insect-resistant transgenic plants were obtained to control plant pests. Vip3A pesticidal protein is one of the numerous pesticidal proteins, which is a specific protein produced by Bacillus cereus.

Vip3A protein has the effect of poisoning and killing sensitive insects by activating

941 701 5_1 (GHMatters) P1 06623.AU programmed death of apoptosis types of cells. Vip3A protein is hydrolysed into four main protein product in the intestinal of the insect, wherein, only one protein hydrolysate (33KD) is the core structure representing the toxicity of Vip3A protein. Vip3A protein binds the epithelial cells of midgut of sensitive insects, thereby to start the programmed death, which can result in dissolution of epithelial cells of midgut so as to cause death of the insects. Vip3A protein does not produce any disease to the non-sensitive insects, so does not result in apoptosis of epithelial cells of midgut and dissolution.

It has been proved that plant genetically modified by Vip3 A may resist the encroachment of Agrotis ypsilon Rottemberg, Spodoptera frugiperda, sesamia inferens, Chrysaspidia festucae and other Lepidoptera pests. However, by now there is no report on controlling harm of Chilo suppressalis to plant through generating transgenic plant expressing Vip3A protein.

Summary of the invention

The present invention is to provide uses of insecticidal protein, particularly provides a method for controlling harm of Chilo suppressalis to plants by producing transgenic plants expressing Vip3A protein, which effectively overcomes the technical limitations of the prior art such as agricultural control, chemical control and physical control.

In order to achieve the purpose mentioned above, the present invention provides following technical solutions, specifically:

The first aspect of the present application relates to a method for controlling Chilo suppressalis, wherein contacting the pest Chilo suppressalis at least with a Vip3A protein;

preferably, the Vip3A protein at least exists in a host cell expressing Vip3A protein, and Chilo suppressalis at least contacts with Vip3A protein by ingestion of the host cell;

more preferably, Vip3A protein at least exists in bacteria or a transgenic plant expressing Vip3A protein, and Chilo suppressalis at least contacts with Vip3A protein by ingestion of the bacteria or a tissue of the transgenic plant; thereafter, the growth of Chilo suppressalis is inhibited and/or Chilo suppressalis dies, so as to achieve controlling over the damage of Chilo suppressalis to the plant.

In further embodiments, as the method for controlling pest Chilo suppressalis, wherein the transgenic plant may be in any growth period; and/or

941 701 5_1 (GHMatters) P1 06623.AU the tissues of the transgenic plant are roots, leaves, stems, fruits, tassels, ears, anthers or filaments; and/or the control of the damage of Chilo suppressalis to the plant does not depond on planting location and/or planting time.

Further, as said method for controlling pest Chilo suppressalis, the plant is selected from the group consisting of rice, sugar cane, wild rice, corn, sorghum, soybean, rape, wheat, millet or barnyard.

Further, as said method for controlling Chilo suppressalis, prior to contacting, the method includes a step of planting a seedling containing a polynucleotide encoding the Vip3A protein.

Further, as said method for controlling Chilo suppressalis, the amino acid sequence of the Vip3A is shown by SEQ ID NO: 1 or SEQ ID NO:3.

Preferably, the nucleotide sequence encoding the Vip3A protein comprises a nucleotide sequence shown by SEQ ID NO:2 or SEQ ID NO:4.

Further, as said method for controlling Chilo suppressalis, the plant also contains at least a second nucleotide sequence which is different from the nucleotide sequence encoding Vip3A protein;

Preferably, the second nucleotide sequence encodes a Cry-type insecticidal protein, a Vip-type insecticidal protein, a protease inhibitor, lectin, α-amylase, peroxidase or a dsRNA inhibiting an important gene of target insect or pest;

more preferably, the second nucleotide sequence encodes CrylAb protein;

still preferably, the amino acid sequence of CrylAb protein comprises an amino acid sequence shown by SEQ ID NO:5;

most preferably, the second nucleotide sequence comprises a nucleotide sequence shown by SEQ ID NO:6.

Further, as the method for controlling Chilo suppressalis, Chilo suppressalis is in the crop field.

On the other hand, the present invention provides use of Vip3A protein to control Chilo suppressalis.

And on another hand, the present invention provides use of a plant cells, a plant tissue, a

941 701 5_1 (GHMatters) P1 06623.AU plant or a bacteria which are transformed with Vip3A gene to control Chilo suppressalis. Again on the other hand, the present invention provides a method for producing a plant that controls Chilo suppressalis, the method includes a step of introducing a polynucleotide sequence encoding Vip3A protein into the genome of the plant.

Also on the one hand, the present invention provides a method for producing a propagule which controls Chilo suppressalis, the method includes a step of hybridizing a first plant produced by the method according to said methods with a second plant and/or the tissue having the reproductive capacity on the plants obtained by said method was removed and cultured, thus produces a propagule containing a polynucleotide sequence encoding Vip3A protein.

On the other hand, the present invention provides a propagule that controls Chilo suppressalis cultured by said methods.

And on another hand, the present invention provides a method for cultivating a plant that controls Chilo suppressalis, the method includes the following steps:

planting at least one propagule whose genome contains a polynucleotide sequence encoding Vip3A protein;

making the propagule grow into a plant;

making the plant grow under the condition of artificial inoculation and/or natural occurrence of Chilo suppressalis, and harvesting the plant with weakened plant damage and/or increased plant yield compared to other plant without the polynucleotide sequence encoding Vip3A protein.

Again on the other hand, the present invention provides a plant that controls Chilo suppressalis cultivated by said methods.

Also on the one hand, the present invention provides a propagule, a plant cell, a plant tissue, a plant or a bacteria that controls Chilo suppressalis, wherein the genome of the propagule, the plant cell, the plant tissue, the plant or the bacteria contains the polynucleotide sequence encoding Vip3A protein;

preferably, the amino acid sequence of Vip3A protein comprises an amino acid sequence shown by SEQ ID NO: 1 or SEQ ID NO:3;

more preferably, the nucleotide sequence encoding Vip3A protein comprises a nucleotide sequence shown by SEQ ID NO:2 or SEQ ID NO:4.

Further, as said propagule, plant cell, plant tissue, plant or bacteria that controls Chilo suppressalis, wherein the genome of the propagule, the plant cell, the plant tissue, the plant or the bacteria at least contains a second nucleotide sequence which is different from the

941 701 5_1 (GHMatters) P106623AU nucleotide sequence encoding Vip3A protein;

preferably, the second polynucleotide encodes a Cry-type insecticidal protein, a Vip-type insecticidal protein, a protease inhibitor, lectin, α-amylase, peroxidase or a dsRNA inhibiting an important gene of target insect or pest;

more preferably, the second polynucleotide sequence encodes CrylAb protein;

still preferably, the amino acid sequence of CrylAb protein comprises an amino acid sequence shown by SEQ ID NO:5;

most preferably, the second nucleotide sequence comprises a nucleotide sequence shown by SEQ ID NO:6.

Further, as said propagule, plant cell, plant tissue, plant or bacteria that controls Chilo suppressalis, the plant is selected from the group consisting of rice, sugar cane, wild rice, corn, sorghum, soybean, rape, wheat, millet or barnyard grass, and the tissues of the transgenic plant are roots, leaves, stems, fruits, tassels, ears, anthers or filaments.

The “propagules” in the present application includes but not limited to plant sexual reproduction and plant asexual propagules. The plant sexual propagules includes but are not limited to plant seeds; the plant asexual propagules refers to vegetative organs of a plant or particular tissue, which can produce new plants in vitro; the vegetative organs or certain special tissues include but are not limited to roots, stems and leaves, for example, plant take root as vegetative propagules includes strawberry and sweet potato,etc; plant take stem as vegetative propagules includes sugarcane and potato (tuber),etc; plant take leaf as vegetative propagules includes vera and begonia,etc.

The “contact” in the present application refers to that insects and/or pests touch, stay and/or intake plants, plant organs, plant tissues or plant cells. The plants, plant organs, plant tissues or plant cells refer to they can expresse insecticidal proteins in vivo or the surface of the plants, plant organs, plant tissues or plant cells has insecticidal proteins or microorganisms generating insecticidal proteins.

Terms “control” and/or “prophylaxis” in the present application refers to that pest Chilo suppressalis at least contact with Vip3A protein, and after the contact, the growth of pest Chilo suppressalis is inhibited and/or the pest Chilo suppressalis dies. Further more, the pest Chilo suppressalis at least contacts with the Vip3A protein through intaking plant tissue, after the contact, the growth of all or some of the pest Chilo suppressalis is inhibited and/or all or some of them die. Inhibition refers to sub-lethality, i.e.: it does not refer to lethal, but it may arouse certain effects in such aspects such as growth and development, behavior, physiology, biochemistry and tissues, for example: the growth and development

941 701 5_1 (GHMatters) P1 06623.AU is slow and/or stops. Meanwhile, the plant should be normal in morphology and can be cultured under conventional methods in order to use them for consumption and/or generation of products. Besides, compared with non-transgenic wild plant, the plant and/or propagule controlling pest Chilo suppressalis which contain polynucleotide sequence encoding Vip3A protein have weakened plant damage under the condition that the pest Chilo suppressalis does harm through artificial inoculation and/or natural occurrence. The concrete manifestation includes but without limitation: improved stalk resistance, and/or increased grain weight and/or yield. The control” and/or “prevent” function of Vip3A protein over the pest Chilo suppressalis may exist independently and will not abate and/or disappear due to the existence of other substances which can “control” and/or “prevent” pest Chilo suppressalis. Specifically, if the tissues of transgenic plant (containing polynucleotide sequence encoding Vip3A protein) simultaneously and/or asynchronously contain and/or generate Vip3A protein and/or another substance which can control pest Chilo suppressalis, then the existence of another substance will neither affect the “control” and/or “prevent” function of Vip3A protein over Chilo suppressalis, nor can result in that the “control” and/or “prevent” function is realized completely by another substance, while irrelevance with Vip3A protein. Under normal conditions, on farmland, the ingestion process of plant tissues by pest Chilo suppressalis is short and can hardly be observed by naked eyes, therefore, under the condition that pest Chilo suppressalis does harm through artificial inoculation and/or natural occurrence, if any tissue of transgenic plant (containing polynucleotide sequence encoding Vip3A protein) has dead pest Chilo suppressalis, and/or pest Chilo suppressalis whose growth is inhibited stays on them, and/or plant damage is weakened compared with non-transgenic wild plant, it means the method and use of the present application are realized, i.e.: the method and/or use for controlling the pest Chilo suppressalis is realised through the contact between pest Chilo suppressalis and Vip3A protein.

In present invention, the Vip3A protein is expressed in a transgenic plant accompanied by the expressions of one or more Cry-class insecticidal proteins and/or Vip-class insecticidal proteins. This co-expression of more than one kind of insecticidal toxins in a same transgenic plant can be achieved by transfecting and expressing the genes of interest in plants by genetic engineering. In addition, Vip3A protein can be expressed in one plant (Parent 1) through genetic engineering operations and Cry-class insecticidal protein and/or Vip-class insecticidal proteins can be expressed in the second plant (Parent 2) through genetic engineering operation. The progeny expressing all genes of Parent 1 and Parent 2

9417015_1 (GHMatters) P1 06623.AU can be obtained by crossing Parent 1 and Parent 2.

RNA interference (RNAi) refers to a highly conserved and effective degradation of specific homologous mRNA induced by double-stranded RNA (dsRNA) during evolution.

Therefore RNAi technology is applied to specifically knock out or shut down the expression of a specific gene of the target insect pest in present invention.

In the classification system, generally, Lepidoptera is divided into suborder, superfamily, family etc according to morphological characteristics such as the neuration of adult wing, chain mode and antenna type, and the Pyralidae is the most abundant species of Lepidoptera, more than 10,000 species of insects have been found around the world, merely China has thousands of records. Most of Pyralidae pests are agricultural pests, lots of them do harm by drilling into stem, like rice borers and Chilo sacchariphagus. Although Chilo suppressalis and rice borers, Chilo sacchariphagus are all belong to Lepidoptera, except the similarity in the classification criteria, there are significant differences in other morphological structures, it is akin to strawberries and apples in plants (both belong to Rosaceae Rosaceae), they have characteristics of flowers bisexual, radiation symmetry, 5 petals and so on, but they have tremendous differences in fruits and plant morphology. Chilo suppressalis has its unique characteristics form morphological point of view when it’s larval or adult.

The insects of Pyralidae were not only different in morphological characteristics, but also differed in feeding habits. For example, Tryporyza incertulas, which also belongs to Pyralidae, only harm rice, is a single food pest. But Chilo suppressalis not only damages rice, but also damages wild rice and sugarcane. Differences in feeding habits also suggest that the enzyme and the receptor protein produced by the digestive system are different. The enzymes produced by the digestive tract are the key point of the B.t. gene effection, the only way to make one certain B. t. gene resist insect, is that the insect has the enzyme or receptor protein which can bind with the certain B. t. protein. More and more reports show that, insects in different family of same order, and even in different species of the same family, have different sensitivity to the same B.t. gene. For example, Vip3A protein shows insect-resistance activity to Ostrinia furnacalis which belongs to Pyralidae, but do not have insect resistant effect on Plodia interpunctella and Ostrinia nubilalis which also belong to Pyralidae. The three pests above all belong to the Lepidoptera, Pyralidae, but the same Bt protein shows different resistance to the three species of Pyralidae pests. In particular, European corn borers and Asian corn borers are even classified to Pyralidae in same genus Ostrinia (same order, same family, same genus), but their responses to the same Bt protein

941 701 5_1 (GHMatters) P1 06623.AU is very different, and thus fully illustrates the interaction between the Bt protein and enzymes and receptors in insects is complex and unpredictable.

The genome of the plants, the plant tissues or the plant cells described in the present invention, refers to any genetic material in the plants, the plant tissues, or the plant cells, including the nucleus, plastids and the genome of mitochondrial.

As described in the present invention, polynucleotides and/or nucleotides form a complete “gene”, encoding proteins or polypeptides in the host cells of interest. It is easy for one skilled in the art to realize that polynucleotides and/or nucleotides in the present invention can be introduced under the control of the regulatory sequences of the target host.

As well known by one skilled in the art, DNA exists typically as double strands, which are complementary with each other. When DNA is replicated in plants, other complementary strands of DNA are also generated. Therefore, the polynucleotides exemplified in the sequence listing and complementary strands thereof are comprised in this invention. The “encoding strand” generally used in the art refers to a strand binding with an antisense strand. For protein expression in vivo, one of the DNA strands is typically transcribed into a complementary strand of mRNA, which serves as the template of protein expression. Actually, mRNA is transcribed from the “antisense” strand of DNA. “Sense strand” or “encoding strand” contains a series of codons (codon is a triplet of nucleotides that codes for a specific amino acid), which might be read as open reading frames (ORF) corresponding to genes that encode target proteins or peptides. RNA which is functionally equivalent with the exemplified DNA was also contemplated in this invention.

Nucleic acid molecule or fragments thereof were hybridized with the present application Vip3A pesticidal gene under stringency condition in this invention. Any regular methods of nucleic acid hybridization or amplification can be used to identify the existence of the pesticidal gene in present invention. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing with other nucleic acid molecules under certain conditions. In present invention, if two nucleic acid molecules can form an antiparallel nucleic acid structure with double strands, it can be determined that these two molecules can hybridize with each other specifically. If two nucleic acid molecules are completely complementary, one of two molecules is called as the “complement” of another one. in this invention, when every nucleotide of a nucleic acid molecule is complementary with the corresponding nucleotide of another nucleic acid molecule, it is identified the two molecules are “completely complementary”. If two nucleic acid molecules can hybridize with each other so that they can anneal to and bind to each other with enough

941 701 5_1 (GHMatters) P1 06623.AU stability under at least normal “low-stringency” conditions, these two nucleic acids are identified as “minimum complementary”. Similarly, if two nucleic acid molecules can hybridize with each other so that they can anneal to and bind to each other with enough stability under normal “high-stringency” conditions, it is identified that these two nucleic acids are “complementary”. Deviation from “completely complementary” can be allowed, as long as the deviation does not completely prevent the two molecules to form a double-strand structure. A nucleic acid molecule which can be taken as a primer or a probe must have sufficiently complementary sequences to form a stable double-strand structure in the specific solvent at a specific salt concentration.

In this invention, basically homologous sequence refers to a nucleic acid molecule, which can specifically hybridize with the complementary strand of another matched nucleic acid molecule under “high-stringency” condition. The stringency conditions for DNA hybridization are well-known to one skilled in the art, such as treatment with 6.0*sodium chloride/sodium citrate (SSC) solution at about 45 °C and washing with 2.0*SSC at 50 °C. For example, the salt concentration in the washing step is selected from 2.0*SSC and 50 °C for the “low-stringency” conditions and 0.2*SSC and 50 °C for the “high-stringency” conditions. In addition, the temperature in the washing step ranges from 22 °C for the “low-stringency” conditions to 65 °C for the “high-stringency” conditions. Both temperature and the salt concentration can vary together or only one of these two variables varies. Preferably, the stringency condition used in this invention might be as below. SEQ ID NO: 1 is specifically hybridized in 6.0*SSC and 0.5% SDS solution at 65 °C. Then the membrane was washed one time in 2*SSC and 0.1% SDS solution and 1*SSC and 0.1% SDS solution, respectively.

Therefore, the insect-resistant sequences which can hybridize with SEQ ID NO: 2 under stringency conditions were comprised in this invention. These sequences were at least about 40%-50% homologous or about 60%, 65% or 70% homologous, even at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homologous to the sequences of present invention.

Genes and proteins described in the present invention not only include the specifically exemplified sequences, but also include parts and/or fragments (including deletion(s) in and/or at the end of the full-length protein), variants, mutants, substitutes (proteins containing substituted amino acid(s)), chimeras and fusion proteins retaining the pesticidal activity thereof. The “variants” or “variation” refers to the nucleotide sequences encoding the same one protein or encoding an equivalent protein having pesticidal activity. The “equivalent protein” refers to the claimed proteins or that have the same or the substantially same bioactivity of anti-CAz/o io

9417015_1 (GHMatters) P1 06623.AU suppressalis.

The “fragment” or “truncation” of the DNA or protein sequences as described in this invention refers to a part or an artificially modified form thereof (e.g., sequences suitable for plant expression) of the original DNA or protein sequences (nucleotides or amino acids) involved in present invention. The sequence length of said sequence is variable, but it is long enough to ensure that the (encoded) protein is an insect toxin.

It is easy to modify genes and to construct genetic mutants by using standard techniques, such as the well-known point mutation technique. Another example method is that described in the U. S. patent 5605793 of randomly splitting DNA and then reassembling them to create other diverse molecules. Commercially available endonucleases can be used to make gene fragments of full-length gene, and exonuclease can also be operated following the standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to remove nucleotides systematically from the ends of these genes. Various restriction enzymes can also be applied to obtain genes encoding active fragments. In addition, active fragments of these toxins can be obtained directly using the proteases.

In the present invention, the equivalent proteins and/or genes encoding these proteins could be derived from B.t. isolates and/or DNA libraries. There are many ways to obtain the pesticidal proteins of the invention. For example, the antibodies raised specifically against the pesticidal protein disclosed and protected in present invention can be used to identify and isolate other proteins from protein mixtures. In particular, the antibody may be raised against the most constant part of the protein and the most different part from other B.t. proteins. These antibodies then can be used to specifically identify equivalent proteins with the characteristic activity using methods of immunoprecipitation, enzyme linked immunosorbent assay (EFISA) or Western blotting assay. It is easy to prepare the antibodies against the proteins, equivalent proteins or the protein fragments disclosed in the present invention using standard procedures in this art. The genes encoding these proteins then can be obtained from microorganisms.

Due to redundancy of the genetic codons, a variety of different DNA sequences can encode one same amino acid sequence. It is available for one skilled in the art to achieve substitutive DNA sequences encoding one same or substantially same protein. These different DNA sequences are comprised in this invention. The “substantially same protein refers to a sequence in which certain amino acids are substituted, deleted, added or inserted but pesticidal activity thereof is not substantially affected, and also includes the fragments remaining the pesticidal activity.

Substitution, deletion or addition of some amino acids in amino acid sequences in this invention

941 701 5_1 (GHMatters) P1 06623.AU is conventional technique in the art. Preferably, such an amino acid change includes: minor characteristics change, i.e. substitution of reserved amino acids which does not significantly influence the folding and/or activity of the protein; short deletion, usually a deletion of about

1-30 amino acids; short elongation of amino or carboxyl terminal, such as a methionine residue elongation at amino terminal; short connecting peptide, such as about 20-25 residues in length.

The examples of conservative substitution are the substitutions happening in the following amino acids groups: basic amino acids (such as arginine, lysine and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (e.g., glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (e.g., phenylalanine, tryptophan and tyrosine), and small molecular amino acids (such as glycine, alanine, serine and threonine and methionine). Amino acid substitutions generally not changing specific activity are well known in the art and have been already described in, for example, “Protein” edited by N. Neurath and R. L. Hill, published by Academic Press, New York in 1979. The most common substitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/ Glu and Asp/Gly, and reverse substitutions thereof.

Obviously, for one skilled in the art, such a substitution may happen outside of the regions which are important to the molecular function and still cause the production of active polypeptides. For the polypeptide of the present invention, the amino acid residues which are required for their activity and chosen as the unsubstituted residues can be identified according to the known methods of the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g. Cunningham and Wells, 1989, Science 244:1081-1085). The latter technique is carried out by introducing mutations in every positively charged residue in the molecule and detecting the insect-resistant activity of the obtained mutation molecules so as to identify the amino acid residues which are important to the activity of the molecules. Enzyme-substrates interaction sites can also be determined by analyzing its three-dimensional structure, which can be determined through some techniques such as nuclear magnetic resonance (NMR) analysis, crystallography, or photoaffinity labeling (see, for example, de Vos et al., 1992, Science 255:306-312,; Smith, et al., 1992, J. Mol. Biol 224:899-904; Wlodaver et al., 1992, FEBS Letters 309:59-64).

In the invention, Vip3A protein includes but is not limited to SEQ ID NO:1, amino acid sequences which have certain homology with the amino acid sequences set forth in SEQ ID NO:

are also comprised in this invention. The sequence similarity/homology between these sequences and the sequences described in the present invention are typically more than 78%, preferably more than 85%, more preferably more than 90%, even more preferably more than

941 701 5_1 (GHMatters) P1 06623.AU

95% and more preferably more than 99%. The preferred polynucleotides and proteins in the present invention can also be defined according to more specific ranges of the homology and/or similarity. For example, they have a homology and/or similarity of 78%, 79%, 80%, 81%, 82%,

83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or

99% with the sequences described in this invention.

In the present invention, the transgenic plant that produces the Vip3A protein includes but is not limited to a COT 102 transgenic cotton event and/or plant material comprising a COT 102 transgenic cotton event (as described in CN1004395507C), a COT202 transgenic cotton event and/or plant material comprising a COT202 transgenic cotton event (as described in CN1886513A), or a MIR162 transgenic maize event and/or plant material comprising a MIR162 transgenic cotton event (as described in CN10154801 IA), which are capable of carrying out the methods and/or use, i.e., by contacting the pest Chilo suppressalis with at least the Vip3A protein to achieve the method and/or use of controlling the pest Chilo suppressalis, and more specifically, the Vip3A protein is exist in a transgenic plant that produces at least the Vip3A protein, the pest Chilo suppressalis is controlled by at least contacting with the Vip3A protein by ingesting the tissue of the transgenic plant, and the growth of the Chilo suppressalis is inhibited and/or leading to death in order to achieve the control of the Chilo suppressalis harm the plants.

Regulatory sequences described in this invention include but are not limited to a promoter, transit peptide, terminator, enhancer, leading sequence, introns and other regulatory sequences that can be operably linked to the pesticidal gene.

The promoter is a promoter expressible in plants, wherein said “a promoter expressible in plants” refers to a promoter which ensures that the encoding sequences bound with the promoter can be expressed in plant cells. The promoter expressible in plants can be a constitutive promoter. The examples of promoters capable of directing the constitutive expression in plants include but are not limited to 35S promoter derived from Cauliflower mosaic virus, Ubi promoter, promoter of GOS2 gene derived from rice and the like. Alternatively, the promoter expressible in plants can be a tissue-specific promoter, which means that the expression level directed by this promoter in some plant tissues such as in chlorenchyma, is higher than that in other tissues of the plant (can be measured through the conventional RNA test), such as the PEP carboxylase promoter. Alternatively, the promoter expressible in plants can be wound-inducible promoters as well. Wound-inducible promoters or promoters that direct wound-inducible expression manners refer to the promoters by which the expression level of the encoding sequences can be increased remarkably compared with those under the normal growth conditions when the plants are

941 701 5_1 (GHMatters) P1 06623.AU subjected to mechanical wound or wound caused by the gnaw of an insect. The examples of wound-inducible promoters include but are not limited to the promoters of genes of protease inhibitor of potato and tomato (pin I and pin II) and the promoter of maize proteinase inhibitor gene (MPI).

The transit peptide (also called secretary signal sequence or leader sequence) directs the gene products into specific organelles or cellular compartment. For the receptor protein, the transit peptide can be heterogeneous. For example, sequences encoding chloroplast transit peptide are used to lead to chloroplast; or ‘KDEL’ reserved sequence is used to lead to the endoplasmic reticulum or CTPP of the barley lectin gene is used to lead to the vacuole.

The leader sequences include but are not limited to small RNA virus leader sequences, such as EMCV leader sequence (encephalomyocarditis virus 5’ non encoding region); Potato virus Y leader sequences, such as MDMV (maize dwarf Mosaic virus) leader sequence; human immunoglobulin heavy chain binding protein (BiP); untranslated leader sequence of the coat protein mRNA of Alfalfa Mosaic virus (AMV RNA4); Tobacco Mosaic virus (TMV) leader sequence.

The enhancer includes but is not limited to Cauliflower Mosaic virus (CaMV) enhancer, Figwort mosaic virus (FMV) enhancer, carnations etched ring virus (CERV) enhancer, cassava vein Mosaic virus (CsVMV) enhancer, mirabilis mosaic virus (MMV) enhancer, Cestrum yellow leaf curling virus (CmYLCV) enhancer, Cotton leaf curl Multan virus (CLCuMV), Commelina yellow mottle virus (CoYMV) and peanut chlorotic streak caulimovirus (PCLSV) enhancer.

For the application of monocotyledon, the introns include but are limited to maize hsp70 introns, maize ubiquitin introns, Adh intron 1, sucrose synthase introns or rice Actl introns. For the application of dicotyledonous plants, the introns include but are not limited to CAT-1 introns, pKANNIBAL introns, PIV2 introns and “super ubiquitin” introns.

The terminators can be the proper polyadenylation signal sequences playing a role in plants. They include but are not limited to polyadenylation signal sequence derived from Agrobacterium tumefaciens nopaline synthetase (NOS) gene, polyadenylation signal sequence derived from protease inhibitor II (pin II) gene, polyadenylation signal sequence derived from peas ssRUBISCO E9 gene and polyadenylation signal sequence derived from α-tubulin gene.

The term “operably linked” described in this invention refers to the linking of nucleic acid sequences, which provides the sequences the required function of the linked sequences. The term “operably linked” described in this invention can be to link a promoter with the sequences of

941 701 5_1 (GHMatters) P1 06623.AU interest, which makes the transcription of these sequences under the control and regulation of the promoter. When the sequence of interest encodes a protein and the expression of this protein is required, the term “operably linked” indicates that the linking of the promoter and said sequence makes the obtained transcript to be effectively translated. If the linking of the promoter and the encoding sequence results in transcription fusion and the expression of the encoding protein are required, such a linking is generated to make sure that the first translation initiation codon of the obtained transcript is the initiation codon of the encoding sequence. Alternatively, if the linking of the promoter and the encoding sequence results in translation fusion and the expression of the encoding protein is required, such a linking is generated to make sure that the first translation initiation codon of the 5’ untranslated sequence is linked with the promoter, and such a linking way makes the relationship between the obtained translation products and the open reading frame encoding the protein of interest meet the reading frame. Nucleic acid sequences that can be operably linked include but are not limited to sequences providing the function of gene expression (i.e. gene expression elements , such as a promoter, 5’untranslated region, introns, protein-encoding region, 3’ untranslated region, polyadenylation sites and/or transcription terminators); sequences providing the function of DNA transfer and/or integration (i.e., T-DNA boundary sequences, recognition sites of site-specific recombinant enzyme, integrase recognition sites); sequences providing selectable function (i.e., antibiotic resistance markers, biosynthetic genes); sequences providing the function of scoring markers; sequences assistant with the operation of sequences in vitro or in vivo (polylinker sequences, site-specific recombinant sequences) and sequences providing replication function (i.e. origins of replication of bacteria, autonomously replicating sequences, centromeric sequences).

The term “pesticidal” or “insect resistance” described in this invention means it is poisonous to crop pests, in order to “control” and/or “prophylaxis” crop pests. Preferably, said “pesticidal” or “insect resistance” means killing crop pests. More specifically, the target insect is pest Chilo suppressalis.

Vip3A protein of this invention is poisonous to most pests of Chilo suppressalis. The plants mentioned in the invention, especially the rice, sugarcane and com, contain exogenous DNA in their genome. The exogenous DNA contains Vip3A gene sequenc, pest Chilo suppressalis is inhibited and/or leaded to death when it contacts with the protein through intaking plant tissues. Inhibition refers to lethal or sub-lethality. Meanwhile, the plant should be normal in morphology and can be cultured under conventional methods in order to use them for consumption and/or generation of products. In addition, the requirement of chemical or biological pesticides of the plant can be essentially eliminated (the chemical or biological

941 701 5_1 (GHMatters) P1 06623.AU pesticides are the ones against pest Chilo suppressalis targeted by the protein encoded by Vip3A gene).

The expression level of pesticidal crystal proteins (ICP) in the plant materials can be determined using various methods described in this field, such as the method of quantifying mRNA encoding the pesticidal protein in the tissue through using specific primers, or the method of quantifying the pesticidal protein directly and specifically.

The pesticidal effect of ICP in the plants can be detected by using different tests. The target insects of the present invention are mainly pests of Chilo suppressalis.

In the present application, the Vip3A protein may have the amino acid sequence shown by SEQ ID NO:1 in the sequence listing. In addition to including the encoding region of Vip3A protein, other elements may also be included, such as: the elements encoding the selective labeled protein.

Further, the expression cassette containing the nucleotide sequence encoding Vip3A protein of the present application may also be expressed together with at least one gene encoding herbicide resistance protein. The herbicide resistance gene includes without limitation: phosphinothricin resistance gene (such as: bar gene and pat gene), phenmedipham resistance gene (such as: pmph gene), glyphosate resistance gene (such as: EPSPS gene), bromoxynil resistance gene, sulfonylurea resistance gene, herbicide dalapon resistance gene, cyanamide resistance gene or glutamine synthetase inhibitor (such as: PPT) resistance gene, thereby obtaining transgenic plant with both high insecticidal activity and herbicide resistance.

In the present application, exogenous DNA is introduced into plant. For example, the gene or expression cassette or recombinant vector encoding the Vip3A protein is introduced into plant cells, the conventional transformation methods include without limitation: agrobacterium tumefaciens-mediated transformation, trace emission bombardment, direct ingestion DNA into protoplast, electroporation or silica whisker mediated DNA introduction.

The present invention provides a pesticidal protein and use thereof with the following advantages:

1. Control through internal cause. The prior art controls the harm of pest Chilo suppressalis mainly through external action, i.e.: external cause, for example, agricultural control, chemical control and physical control; while the present application controls pest Chilo suppressalis through generating Vip3A protein which can kill Chilo suppressalis inside the plant, i.e.: controls pest Chilo suppressalis through internal cause.

941 701 5_1 (GHMatters) P1 06623.AU

2. No pollution and no residue. The chemical control method used in prior art plays certain role in controlling the harm of pest Chilo suppressalis, but in the same time, it also causes pollution, destruction and residue to human, livestock and farmland ecosystem; using the method for controlling pest Chilo suppressalis provided in the present application may eliminate the foregoing bad consequences.

3. Control throughout all growth period. All the methods for controlling pest Chilo suppressalis used in prior art are staged, while the present application protects the plant throughout all growth period so that transgenic plant (Vip3A protein) can be free from the encroachment of Chilo suppressalis from sprouting, growth and till blooming and fruit.

4. Control over whole plant. The method for controlling pest Chilo suppressalis used in the prior art mostly are localised, for example foliage spray; while the present application protects whole plant, for example, the roots, leaves, stalks, fruits, tassels, pistils, anthers or filaments and so on of transgenic plant (Vip3A protein) all may resist the harm of Chilo suppressalis.

5. Effect stability. Used in the prior art, the dirt on the the high-voltage grid of frequency vibration insecticidal lamp needs to be cleaned up everyday, and the lamp cannot be open in thunderstorm days; in the present invention, the Vip3A protein is expressed in vivo in the plant, which effectively overcomes the defects of the effect of the frequency vibration type insecticidal lamp affected by external factors, and the control effect of the transgenic plant (Vip3A protein) of the present invention is consistence in different locations, different times , different genetic backgrounds.

6. Simple, convenient and economical. The frequency vibration insecticidal lamp used in prior art has a large one-time cost in application, and under the risk of hurting people by electric shork in operation; while the present application only needs to plant the transgenic plant which can express Vip3A protein and does not need to take other measures, thereby saving a large amount of human, material and financial resources.

7. Thorough effect. The method for controlling pest Chilo suppressalis used in the prior art does not have a thorough effect and only plays a role of mitigation, while transgenic plant (Vip3A protein) of the present invention has almost 100% control effect on the newly hatched larvae of Chilo suppressalis, very few surviving larvae are basically stopped developing, after 3 days, larvae are still in newly-hatched state, obviously dysplasia, and has stopped developing, can not survive in the natural environment, while transgenic plants only suffer minor damage in general.

941 701 5_1 (GHMatters) P1 06623.AU

The technical solutions of this invention will be further described through the appended figures and examples as following.

Brief description of the drawings

Figure 1 shows the scheme to construct the recombinant cloning vector DBN01-T containing Vip3A nucleotide sequence for uses of insecticidal protein in this invention;

Figure 2 shows the scheme to construct the recombinant expression vector DBN100002 containing Vip3A nucleotide sequence for uses of insecticidal protein in this invention;

Figure 3 shows the leaf damage of transgenic rice plants for uses of insecticidal protein against Chilo suppressalis in this invention.

Detailed description of the invention

The technical solutions of this invention for uses of insecticidal protein will be further illustrated through the following examples.

Example 1: The obtaining and synthesis of gene

1. Obtaining of nucleotide sequence

Amino acid sequence of Vip3A-01 pesticidal protein (789 amino acids) was shown as SEQ ID NO:1 in the sequence listing; the nucleotide sequence of Vip3A-01 gene (2370 nucleotides) encoding the corresponding amino acid sequence of Vip3A-01 pesticidal protein was shown as SEQ ID NO:2 in the sequence listing.

Amino acid sequence of Vip3A-02 pesticidal protein (789 amino acids) was shown as SEQ ID NO:3 in the sequence listing; the nucleotide sequence of Vip3A-02 gene (2370 nucleotides) encoding the corresponding amino acid sequence of Vip3A-02 pesticidal protein was shown as SEQ ID NO:4 in the sequence listing.

Amino acid sequence of CrylAb pesticidal protein (615 amino acids) was shown as SEQ ID NO:5 in the sequence listing; the nucleotide sequence of CrylAb gene (1848 nucleotides) encoding the corresponding amino acid sequence of CrylAb pesticidal protein was shown as SEQ ID NO:6 in the sequence listing.

2. Synthesis of the nucleotide sequence as described above

The Vip3A-01 nucleotide sequence (shown as SEQ ID NO:2 in the sequence listing), the

941 701 5_1 (GHMatters) P1 06623.AU

Vip3A-02 nucleotide sequence (shown as SEQ ID NO:4 in the sequence listing) and the CrylAb nucleotide sequence (shown as SEQ ID NO:6 in the sequence listing) were synthesized by GenScript CO., LTD, Nanjing; The synthesized Vip3A-01 nucleotide sequence (SEQ ID NO:2) was linked with a Seal restriction site at the 5’ end and a Spel restriction site at the 3’ end; the synthesized Vip3A-02 nucleotide sequence (SEQ ID NO:4) was linked with a Seal restriction site at the 5’ end and a Spel restriction site at the 3’ end; the synthesized Cryl Ab nucleotide sequence (SEQ ID NO:6) was linked with an Ncol restriction site at the 5’ end and a BamHI restriction site at the 3 ’ end.

Example 2: Construction of recombinant expression vectors and the transfection of Agrobacterium with the recombinant expression vectors

1. Construction of the recombinant cloning vectors containing Vip3A gene

The synthesized Vip3A-01 nucleotide sequence was sub-cloned into cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), to get cloning vector DBN01-T following the instructions of Promega pGEM-T vector, and the construction process was shown in Figure 1 (wherein the Amp is ampicillin resistance gene; f 1 is the replication origin of phage fl; LacZ is initiation codon of LacZ; SP6 is the promoter of SP6 RNA polymerase; T7 is the promoter of T7 RNA polymerase; Vip3A-01 is Vip3A-01 nucleotide sequence (SEQ ID NO:2); MCS is multiple cloning sites).

The recombinant cloning vector DBN01-T was then transformed into E. coli T1 competent cell (Transgen, Beijing, China, the CAT: CD501) through heat shock method. The heat shock conditions were as follows: 50μΙ of E. coli T1 competent cell and 10μ1 of plasmid DNA (recombinant cloning vector DBN01-T) were incubated in water bath at 42 °C for 30 seconds; then the E. coli cells were incubated in water bath at 37 °C for 1 h (100 rpm in a shaking incubator) and then were grown on a LB plate (lOg/L Tryptone, 5g/L yeast extract, lOg/L NaCl, 15g/L Agar and pH was adjusted to 7.5 with NaOH) coated on the surface with IPTG (Isopropyl thio-beta-D-galactoseglucoside) , X-gal (5-bromine-4-chlorine-3-indole-beta-D-galactose glucoside) and ampicillin (lOOmg/L) overnight. The white colonies were picked out and cultivated in LB broth (lOg/L Tryptone, 5g/L yeast extract, lOg/L NaCl, 100 mg/L ampicillin and pH was adjusted to 7.5 with NaOH) at 37 °C overnight. The plasmids thereof were extracted using alkaline lysis method as follows: the cell broth was centrifuged for 1 min at 12000 rpm, the supernatant was discarded and the pellet was resuspended in 100 μΐ of ice-chilled solution I (25 mM Tris- HC1, 10 mM EDTA (ethylenediaminetetraacetic acid) and 50 mM glucose, pH 8.0); then 200μ1 of freshly prepared solution II (0.2 M NaOH, 1% SDS (sodium dodecyl sulfate)) was

941 701 5_1 (GHMallers) P106623AU added and the tube was reversed 4 times, mixed and then put on ice for 3-5 min; 150μ1 of cold solution III (3 M potassium acetate and 5 M acetic acid) was added, thoroughly mixed immediately and incubated on ice for 5-10 min; the mixture was centrifuged at 12000 rpm at 4 °C for 5 min, two volumes of anhydrous ethanol were added into the supernatant, mixed and then placed at room temperature for 5 min; the mixture was centrifuged at 12000 rpm at 4 °C for 5 min, the supernatant was discarded and the pellet was dried after washed with 70% ethanol (V/V); 30 μΐ TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing RNase (20pg /ml) was added to dissolve the precipitate; the mixture was incubated at 37 °C in a water bath for 30 min to digest RNA and stored at - 20 °C for the future use.

After the extracted plasmids were confirmed with restriction enzymes AhdI and Xhol, the positive clones were verified through sequencing. The results showed that the Vip3A-01 nucleotide sequence inserted into the recombinant cloning vector DBN01-T was the sequence set forth in SEQ ID NO:2 in the sequence listing, indicating that Vip3A-01 nucleotide sequence was correctly inserted.

The synthesized nucleotide sequence Vip3A-02 was inserted into cloning vector pGEM-T to get recombinant cloning vector DBN02-T following the process for constructing cloning vector DBN01-T as described above, wherein Vip3A-02 was Vip3A-02 nucleotide sequence (SEQ ID NO:4). The Vip3A-02 nucleotide sequence in the recombinant cloning vector DBN02-T was verified to be correctly inserted with restriction enzyme digestion and sequencing.

The synthesized nucleotide sequence CrylAb was inserted into cloning vector pGEM-T to get recombinant cloning vector DBN03-T following the process for constructing cloning vector DBN01-T as described above, wherein CrylAb was CrylAb nucleotide sequence (SEQ ID NO:6). The CrylAb nucleotide sequence in the recombinant cloning vector DBN03-T was verified to be correctly inserted with restriction enzyme digestion and sequencing.

2. Construction of the recombinant expression vectors containing Vip3A gene

The recombinant cloning vector DBN01-T and expression vector DBNBC-01 (Vector backbone: pCAMBIA2301, available from CAMBIA institution) were digested with restriction enzymes Seal and Spel, the cleaved Vip3A-01 nucleotide sequence fragment was ligated between the restriction sites Seal and Spel of the expression vector DBNBC-01 to construct the recombinant expression vector DBN100002, it is a well-known conventional method to construct expression vector through restriction enzyme digestion, the construction scheme was shown in Figure 2 (Kan: kanamycin gene; RB: right border; CaMV35S: cauliflower mosaic virus 35S promoter (SEQ ID NO:7); Vip3A-01: Vip3A-01 nucleotide sequence (SEQ ID NO:2); Nos, terminator of

941 701 5_1 (GHMatters) P1 06623.AU nopaline synthetase gene (SEQ ID NO:8); Hpt: hygromycin phosphotransferase gene (SEQ ID NO:9); LB: left border).

The recombinant expression vector DBN100002 was transformed into E. coli TI competent cells with heat shock method as follows: 50μ1 of E. coli TI competent cell and 10μ1 of plasmid DNA (recombinant expression vector DBN 100002) were incubated in water bath at 42 °C for 30 seconds; then the E. coli cells were incubated in water bath at 37 °C for 1 hour (100 rpm in a shaking incubator) and then were grown on a LB solid plate (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 15 g/L Agar and pH was adjusted to 7.5 with NaOH) containing 50 mg/L kanamycin at 37 °C for 12 hours, the white colonies were picked out and cultivated in LB broth (10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 50 mg/L kanamycin and pH was adjusted to 7.5 with NaOH) at 37 °C overnight. The plasmids thereof were extracted using alkaline lysis method. After the extracted plasmids were confirmed with restriction enzymes Seal and Spel, the positive clones were verified through sequencing, the results showed that the nucleotide sequence between restriction sites Seal and Spel in the recombinant expression vector DBN100002 was the nucleotide sequence set forth in SEQ ID NO:2 in the sequence listing, i.e. Vip3A-01 nucleotide sequence.

Following the process for constructing recombinant expression vector DBN 100002 as described above, recombinant cloning vectors DBN02-T and DBN03-T were digested with restriction enzymes Scal/Spel and NcoI/BamHI respectively to cleave the Vip3A-02 nucleotide sequence and Cry 1 Ab nucleotide sequence which then were inserted into the expression vector DBNBC-01 to get the recombinant expression vector DBN 100003. Restriction enzyme digestion and sequencing verified that recombinant expression vector DBN 100003 contained the nucleotide sequences set forth in SEQ ID NO:4 and SEQ ID NO:6 in the sequence listing, i.e. the nucleotide sequences of Vip3A-02 and CrylAb, the nucleotide sequences of Vip3A-02 and Cryl Ab may linked with CaMV35S promoter and Nos terminator.

3. Transfection of Agrobacterium tumefaciens with the recombinant expression vectors

The correctly constructed recombinant expression vectors DBN 100002 and DBN 100003 were transfected into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, Cat. No: 18313-015) following liquid nitrogen rapid-freezing method as follows: 100 pL Agrobacterium LBA4404 and 3 pL plasmid DNA (recombinant expression vector) were put into liquid nitrogen for 10 min and then incubated in water bath at 37 °C for 10 min; then the transfected Agrobacterium LBA4404 cells were inoculated in LB broth and cultivated at 28 °C, 200 rpm for 2 hours and spraid on a LB plate containing 50 mg/L of rifampicin (Rifampicin) and 100 mg/L of kanamycin

941 701 5_1 (GHMatters) P1 06623.AU (Kanamycin) until positive mono colonies appeared, the positive mono colonies were picked up and cultivated and the plasmids thereof were extracted, recombinant expression vectors

DBN100002 and DBN100003 were verified with restriction enzymes Styl and Aatll, the results showed that the recombinant expression vectors DBN 100002 and DBN 100003 were correct in structure, respectively.

Example 3: Obtaining of the transgenic plant

1. Obtaining of the transgenic rice plant

According to the conventional Agrobacterium transfection method, the rice cultivar Nihonbare was cultivated in sterilized conditions and the callus was co-cultivated with the Agrobacterium strains constructed in part 3 of Example 2 so as to introduce T-DNAs in the recombinant expression vectors DBN 100002 and DBN 100003 constructed in part 2 of Example 2 (including cauliflower mosaic virus 35S gene promoter sequence, Vip3A-01 nucleotide sequence, Vip3A-02 nucleotide sequence, CrylAb nucleotide sequence, Hpt gene and Nos terminator sequence) into the rice genome, rice plants containing Vip3A-01 nucleotide sequence and rice plants containing Vip3A-02-CrylAb nucleotide sequence were obtained respectively; wild type rice plant was taken as a control.

Regarding to the Agrobacterium -mediated transfection of rice, briefly, rice seeds were inoculated on induction medium (N6 salt, N6 vitamins, 300 mg/L of casein, 30 g/L of sucrose, 2 mg/L of

2,4-dichlorophenoxyacetic acid (2,4-D) and 3g/L of plant gelatum, pH=5.8) and callus was induced from mature embryo of rice (Step 1: callus induction step). Then the next is to optimize callus. Callus was contacted with Agrobacterium suspension, in which the Agrobacterium can deliver the Vip3A-01 nucleotide sequence, Vip3A-02nucleotide sequence, CrylAb nucleotide sequence into at least one cell of the callus (Step 2: infection step). In this step, preferably, callus was immersed in Agrobacterium suspension (OD₆₆₀ = 0.3, infection medium ( N6 salt, N6 vitamins, 300 mg/L of casein, 30 g/L of sucrose, 10 g/L of glucose, 40 mg/L of Acetosyringone (AS), 2 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D), pH=5.4) to initiate the infection. Callus and Agrobacterium were cocultivated for a period (3 days) (Step 3: cocultivation step). Preferably, callus was cultivated on a solid medium (N6 salt, N6 vitamins, 300 mg/L of casein, 30 g/L of sucrose, 10 g/L of glucose, 40 mg/L of Acetosyringone (AS), 2 mg/L of

2,4-dichlorophenoxyacetic acid (2,4-D) and 3 g/L of plant gelatum, pH=5.8) after the infection step. After this cocultivation step, a recovery step can be proceded. In the recovery step, the recovery medium (N6 salt, N6 vitamins, 300 mg/L of casein, 30 g/L of sucrose, 2 mg/L of

2,4-dichlorophenoxyacetic acid (2,4-D) and 3 g/L of plant gelatum, pH=5.8) contains at least one

941 701 5_1 (GHMatters) P1 06623.AU kind of known Agrobacterium-mhWdmg antibiotics (cephamycin) without the selective agent for plant transfectants (Step 4: recovery step). Preferably, the callus was cultivated on a solid medium culture containing antibiotics but without selective agent so as to eliminate Agrobacterium and to provide a recovery period for the infected cells. Then the inoculated callus was cultivated on a medium containing selective agent (mannose) and the transfected callus was selected (Step 5: selection step). Preferably, the callus was cultivated on a selective solid medium containing selective agent (N6 salt, N6 vitamins, 300 mg/L of casein, 10 g/L of sucrose, 50mg/L of hygromycin, 2 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3 g/L of plant gelatum, pH=5.8), resulting the selective growth of the transfected cells. Then, callus regenerated into plants (Step 6: regeneration step). Preferably, the callus was cultivated on a solid medium containing selective agent (N6 differentiation medium and MS rooting medium) to regenerate into plants.

The obtained resistant callus was transferred to the N6 differentiation medium (N6 salt, N6 vitamins, 300 mg/L of casein, 20 g/L of sucrose, 2 mg/L of 6-benzyladenine, 1 mg /L of naphthylacetic acid and 3 g/L of plant gelatum, pH=5.8) and cultivated and differentiated at 25 °C. The differentiated seedlings were transferred to the MS rooting medium (MS salt, MS vitamins, 300 mg/L of casein, 15 g/L of sucrose, 3 g/L of plant gelatum, pH=5.8) and cultivated to about 10 cm in height at 25 °C. Next, the seedlings were transferred to and cultivated in the greenhouse until fructification. In the greenhouse, the rice plants were cultivated at 30 °C every day.

2. Obtaining of transgenic sugarcane plant

The transformation method is mainly based on the Guangxi University 2012 Master's Degree Li Can's dissertation page 22 to 24. Take the top of sugarcane’s new stem node, remove the cane shoots and leaf sheath, leaving only the stem apical growth cone and heart stems segment. The surface was wiped and disinfected with 75% (v/v) alcohol cotton balls on the clean bench, carefully peelling off the outer layer of the heart leaf with sterilized tweezers, taking the 5-7cm long heart segment in the middle, cutting transversely to about 3mm sheet and inoculated on the induction medium, temperature at 26 ° C under dark conditions for 20 days. Callus which growth well were selected and transferred to the new MS medium for 4 days and then used for the transformation test; when transformating, the callus which are about to be infected is caught out with sterilized tweezer on the clean bench, and placed on a clean filter paper for 2 hours, until the surface is fully dried, slightly contracted; the dried sugarcane callus was immersed in the infusion solution for 30 minutes, at the same time slowly shaked in the shaker; the callus was

941 701 5_1 (GHMatters) P1 06623.AU removed and transferred to clean filter paper and dried thoroughly on a clean bench until the callus surface was dry and with no water film. The callus was cut into small pieces of 0.6 * 0.6 cm, then transferred to an MR solid medium containing 100 μιηοΙ/L acetosyringone (AS), incubated at 23 ° C for 3 days; after the infection, callus were caught out and placed in the filter paper on the clean bench to dry, until the surface of material dry up, the material was transferred to a differentiation medium containing 500 mg/L cephalosporin and hygromycin; replace the medium once every 2 weeks, the contaminated calli were removed during the period, when the seedlings reach 3 cm high, transfer them to the rooting medium which contains hygromycin screening agent to induce rooting. The sugarcane plants transferred with the Vip3A-01 nucleotide sequence and the sugarcane plants transferred wich the Vip3A-02-CrylAb nucleotide sequence were obtained; the wild type sugarcane plants were used as control.

Example 4. Verification of transgenic plants using TaqMan technique

100 mg of leaves from rice plant transfected with Vip3A-01 nucleotide sequence and rice plant transfected with Vip3A-02-CrylAb nucleotide sequence was taken as sample respectively. Genomic DNA thereof was extracted using DNeasy Plant Maxi Kit (Qiagen) and the copy numbers of Vip3A gene and Cry 1 Ab gene were quantified through Taqman probe-based fluorescence quantitative PCR assay. Wild type rice plant was taken as a control and analyzed according to the processes as described above. Experiments were carried out in triplicate and the results were the mean values.

The specific method for detecting the copy numbers of Vip3A gene and CrylAb gene was described as follows.

Step 11: 100 mg of leaves from every transfected rice plant (rice plant transfected with nucleotide sequence of Vip3A-01 and Vip3A-02-CrylAb, respectively) was taken and grinded into homogenate in a mortar in liquid nitrogen respectively. It was in triplicate for each sample.

Step 12: the genomic DNAs of the samples above were extracted using DNeasy Plant Mini Kit (Qiagen) following the product instruction thereof.

Step 13: the genome DNA concentrations of the above samples were determined using NanoDrop 2000 (Thermo Scientific).

Step 14: the genome DNA concentrations were adjusted to the same range of 80-100 ng/μΐ.

Step 15: the copy numbers of the samples were quantified using Taqman probe-based fluorescence quantitative PCR assay, the quantified sample with known copy number was taken as a standard sample and the wild type rice plant was taken as a control. It was carried out in

941 701 5_1 (GHMatters) P1 06623.AU triplicate for every sample and the results were the mean values. Primers and the probes used in the fluorescence quantitative PCR were shown as below.

The following primers and probe were used to detect Vip3A-01 and Vip3A-02 nucleotide sequence:

Primer 1: ATTCTCGAAATCTCCCCTAGCG (as shown in SEQ ID NO: 10 in the sequence listing);

Primer 2: GCTGCCAGTGGATGTCCAG (as shown in SEQ ID NO:11 in the sequence listing);

Probe 1: CTCCTGAGCCCCGAGCTGATTAACACC (as shown in SEQ ID NO: 12 in the sequence listing)

The following primers and probe were used to detect CrylAb nucleotide sequence:

Primer 3: TGCGTATTCAATTCAACGACATG (as shown in SEQ ID NO: 13 in the sequence listing);

Primer 4: CTTGGTAGTTCTGGACTGCGAAC (as shown in SEQ ID NO: 14 in the sequence listing);

Probe 2: CAGCGCCTTGACCACAGCTATCCC (as shown in SEQ ID NO: 15 in the sequence listing);

PCR reaction system was as follows:

JumpStart™ Taq ReadyMix™ (Sigma) 10μ1

OX primer/probe mixture 1 μΐ

Genomic DNA 3μ1

Water (ddH₂O) 6μΙ

The 50X primer/probe mixture contained 45 μΐ of each primer (1 mM), 50 μΐ of probe (ΙΟΟμΜ) and 860 μΐ of 1XTE buffer and was stored in an amber tube at 4 °C.

PCR reaction conditions were provided as follows:

Step	Temperature	Time
21	95 °C	5 min
22	95 °C	30s
23	60 °C	lmin 25

9417015_1 (GHMatters) P1 06623.AU back to step 22 and repeated 40 times

Data were analyzed using software SDS 2.3 (Applied Biosystems).

The experimental results showed that all the nucleotide sequences of Vip3A-01 and Vip3A-02-CrylAb have been integrated into the genomes of the detected rice plants, respectively. Furthermore, rice plants transfected with nucleotide sequences of Vip3A-01 and Vip3A-02-CrylAb respectively contained single copy of Vip3A-01 and Vip3A-02-CrylAb gene respectively.

The transgenic sugarcane plants were tested and analyzed according to the method of testing transgenic rice plants mentioned above.The result shows that all the nucleotide sequences of Vip3A-01 and Vip3A-02-CrylAb have been integrated into the genomes of the detected sugarcane plants, respectively. Furthermore, sugarcane plants transfected with nucleotide sequences of Vip3A-01 and Vip3A-02-CrylAb respectively contained single copy of Vip3A-01 and Vip3A-02-Cryl Ab gene respectively.

Example 5: Detection of pesticidal protein contents in transgenic plants

The rice plants transformed with the Vip3A-01 nucleotide sequence, the rice plants transformed with the Vip3A-02-CrylAb nucleotide sequence; the sugarcane plants transformed with the Vip3A-01 nucleotide sequence; the sugarcane plants transformed with the Vip3A-02-CrylAb nucleotide sequence; corresponding wild-type rice plants and sugarcane plants, as well as non-transgenic rice plants and sugarcane plants which were tested by Taqman are being tested for insect-resistance to Chilo suppressalis.

1. Detecting the insecticidal effect of transgenic rice plants

Fresh leaves are taken from the rice plant transformed with Vip3A-01 nucleotide sequence, the rice plant transformed with Vip3A-02-CrylAb nucleotide sequence, wild rice plant and non-transgenic rice plant as identified by Taqman (tillering stage) respectively and washed with sterile water. The water on the leaves is sucked dry by gauze. Meanwhile the leaves are cut into squares of about lcmx4cm. A cut square leaf is put on the filter paper at the bottom of a round plastic culture disk. The filter paper is moistened with distilled water. 10 artificially fed pest Chilo suppressalis (newly hatched larvae) are put into each culture dish. The culture dishes with pests are covered and then put into a square box with wet gauze at its bottom and rest for 3 days under the conditions of 28 °C, RH 70%-80% and photoperiod (light/dark) 16:8, based on three indexes like development progress, mortality of Chilo suppressalis larvae and leaf damage rate, the total score of resistance is obtained (Total

941 701 5_1 (GHMatters) P1 06623.AU score 300): Total score of resistance=100^xmortality+[100^xmortality+90^x(number of newly hatched larvae/total number of inoculating larvae)+60^x(number of newly hatched larvae number of pests in negative controls/total number of inoculating larvae)+10^x(number of pests in negative controls/total number of inoculating larvae)]+100^x(l-leaf damage rate).

There are 3 strains (SI, S2 and S3) transformed with Vip3A-01 nucleotide sequence, 3 strains (S4, S5 and S6) transformed with Vip3A-02-CrylAb nucleotide sequence, a strain identified by Taqman to be non-transgenic (NGM1) and a wild strain (CK1); three plant are selected from each strain to do test and each plant is tested six times. The results are shown in Table 1 and FIG. 3.

Table 1 Results of insecticidal experiments of transgenic rice plant inoculated with Chilo suppressalis

Plant	Leaf damage rate (%)	Development progress of Death situation ( Chilo suppressalis (single suppressalis	)f Chilo (single	Total score (single strain)	Average total score
strain)	strain)
Newly hatched	Newly hatched - negative control	Snegat ive control	Total number of inoculating larvae	Mortal ity (%)
SI	1	1.5	0	0	10	85	283
S2	5	3.5	0	0	10	65	257	278
S3	1	0.5	0	0	10	95	294
S4	1	0	0	0	10	100	299
S5	1	0	0	0	10	100	299	295
S6	1	1	0	0	10	90	288
NGM1	55	0	0	10	10	0	55	55
CK1	55	0	0	9.5	10	5	65	65

The results in Table 1 indicate: The rice plant transformed with Vip3A-01 nucleotide sequence, the rice plant transformed with Vip3A-02-CrylAb nucleotide sequence both has good insecticidal effect on Chilo suppressalis, the death rate of the Chilo suppressalis are 80% above, some of them can reach 100%, the total scores are around 280 points; the total scores of non-transgenic rice plant identified by Taqman and wild rice plant in bioassay are around 60 points in general.

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The results in FIG. 3 indicate: compared with wild rice plant, the rice plant transformed with Vip3A-01 nucleotide sequence, the rice plant transformed with Vip3A-02-CrylAb nucleotide sequence have almost one hundred percent control effect on the newly hatched Chilo suppressalis. Very few surviving larvae are basically stopped developing. 3 days later, the larvae are basically in a newly hatched state and in the mean time show very weak vitality. Moreover, the rice plant transformed with Vip3A-01 nucleotide sequence, the rice plant transformed with Vip3A-02-CrylAb nucleotide sequence basically suffer mild damage, the feeding traces of Chilo suppressalis are almost impossible to identify by naked eyes, the leaf damage rates are all below 5%.

Thus it is proved that the rice plant transformed with Vip3A-01 nucleotide sequence, the rice plant transformed with Vip3A-02-CrylAb nucleotide sequence both show high activity against Chilo suppressalis and this activity is enough to generate harmful effect on the growth of Chilo suppressalis, so as to control it on farmland. At the same time, by controlling the borer harm of Chilo suppressalis, it is also possible to reduce the occurrence of disease on rice and greatly improve the yield and quality of rice.

2. Detecting the insecticidal effect of transgenic sugarcane plants

Fresh leaves are taken from the sugarcane plant transformed with Vip3A-01 nucleotide sequence, the sugarcane plant transformed with Vip3A-02-CrylAb nucleotide sequence, wild sugarcane plant and non-transgenic sugarcane plant as identified by Taqman (unfolded young leaves) respectively and washed with sterile water, the water on the leaves is sucked dry by gauze, meanwhile the leaves are cut into squares of about lcm^x2cm, a cut square leaf is put on the filter paper at the bottom of a round plastic culture disk. The filter paper is moistened with distilled water. 10 artificially fed pest Chilo suppressalis (newly hatched larvae) are put into each culture dish. The culture dishes with pests are covered and then put into a square box with wet gauze at its bottom and rest for 3 days under the conditions of 22-26°C, RH 70%-80% and photoperiod (light/dark) 16:8. Based on three indexes like development progress, mortality of Chilo suppressalis larvae and leaf damage rate, the total score (total score 300) of resistance is obtained: Total score of risistance=100^xmortality+[100^xmortality+90^x(number of newly hatched larvae/total number of inoculating larvae)+60^x(number of newly hatched larvae - number of pests in negative controls/total number of inoculating larvae)+10^x(number of pests in negative controls/total number of inoculating larvae)]+100^x(l-leaf damage rate). There are 3 strains (S7, S8 and S9) transformed with Vip3A-01 nucleotide sequence, 3 strains (S10, Sil and

S12) transformed with Vip3A-02-CrylAb nucleotide sequence, a strain identified by

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Taqman to be non-transgenic (NGM2) and a wild strain (CK2); three plant are selected from each strain to do test and each plant is tested six times. The results are shown in Table

2.

Table 2 Results of insecticidal experiments of transgenic sugarcane plant inoculated with Chilo 5 suppressalis

Plant	Leaf damage rate (%)	Development progress of Death situation ( Chilo suppressalis (single suppressalis	)f Chilo (single	Total score (single strain)	Average total score
strain)	strain)
Newly hatched	Newly hatched - negative control	Snegat ive control	Total number of inoculating larvae	Mortal ity (%)
S7	3	1.5	0	0	10	85	281
S8	3	1.5	0	0	10	85	281	285
S9	1	0.5	0	0	10	95	294
S10	1	0.5	0	0	10	95	294
sii	1	0.5	0	0	10	95	294	295
S12	1	0	0	0	10	100	299
NGM2	75	0	0	10	10	0	35	35
CK2	65	0	0	9.5	10	5	55	55

The results in Table 2 indicate: The sugarcane plant transformed with Vip3A-01 nucleotide sequence, the sugarcane plant transformed with Vip3A-02-CrylAb nucleotide sequence both have good insecticidal effect on Chilo suppressalis, the death rate of the Chilo suppressalis are

85% above .parts of them can reach 100%, the total scores are above 280 points; The total scores of non-transgenic sugarcane plant identified by Taqman and wild sugarcane plant in bioassay are generally below 55 points.

Compared with wild sugarcane plant, the sugarcane plant transformed with Vip3A-01 nucleotide sequence, the sugarcane plant transformed with Vip3A-02-CrylAb nucleotide sequence have almost one hundred percent control effect on the newly hatched Chilo suppressalis. Very few surviving larvae are basically stopped developing. 3 days later, the larvae are basically in a newly hatched state and in the mean time show very weak vitality. Moreover, the sugarcane

941 701 5_1 (GHMatters) P1 06623.AU plant transformed with Vip3A-01 nucleotide sequence and the sugarcane plant transformed with Vip3A-02-CrylAb nucleotide sequence basically suffer mild damage, the feeding traces of Chilo suppressalis are almost impossible to identify by naked eyes, the leaf damage rates are all below 3%.

Thus it is proved that the sugarcane plant transformed with Vip3A-01 nucleotide sequence, the sugarcane plant transformed with Vip3A-02-CrylAb nucleotide sequence both show high activity against Chilo suppressalis and this activity is enough to generate harmful effect on the growth of Chilo suppressalis, so as to control it on farmland. At the same time, by controlling the borer of Chilo suppressalis, it is also possible to reduce the occurrence of disease on sugarcane and greatly improve the yield and quality of sugarcane.

The above experimental results also indicate that the rice plant transformed with Vip3A-01 nucleotide sequence, the rice plant transformed with Vip3A-02-CrylAb nucleotide sequence, the sugarcane plant transformed with Vip3A-01 nucleotide sequence, the sugarcane plant transformed with Vip3A-02-CrylAb nucleotide sequence can control/prophylaxis Chilo suppressalis apparently because the plant themselves can generate Vip3A protein. Therefore, it is well known to those skilled in the art that based on the same toxic fuction of Vip3A protein to Chilo suppressalis, transgenic plant that may generate similar expressive Vip3A protein can be used to control the harm of Chilo suppressalis. The Vip3A protein in the present application includes without limitation the Vip3A protein given amino acid sequence in embodiments. Meanwhile, the transgenic plant may also generate at least a second-type insecticidal protein different from Vip3A protein, such as Vip3A protein, Cryl A protein.

To summarize, uses of insecticidal protein of the present application controls pest Chilo suppressalis through generating Vip3A protein inside the plant to kill Chilo suppressalis', compared with agricultural control method, chemical control method and physical control method used in the prior art, the present application protects the whole plant throughout all growth period to control encroach of pest Chilo suppressalis, furthermore, it causes no pollution and no residue and provides a stable and thorough control effect. Also it is simple, convenient and economic.

Finally what should be explained is that all the above examples are merely intentioned to illustrate the technical solutions of present invention rather than to restrict present invention. Although detailed description of this invention has been provided by referring to the preferable examples, one skilled in the art should understand that the technical solutions of the invention can be modified or equivalently substituted while still fall within the spirit and scope of the present invention.

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In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense,

i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country

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2016228052 08 Apr 2019

Claims (15)

1. A method for controlling Chilo suppressalis, wherein the method includes contacting Chilo suppressalis at least with Vip3A protein, which comprises an amino acid sequence

5 shown in SEQ ID NO: 1 or SEQ ID NO:3.

2. A method according to claim 1, wherein the Vip3A protein at least exists in a host cell expressing Vip3A protein, and Chilo suppressalis at least contacts with Vip3A protein by ingestion of the host cell.

3. A method according to claim 2, wherein the Vip3A protein at least exists in bacteria or 10 transgenic plant expressing Vip3A protein, and Chilo suppressalis at least contacts with

Vip3A protein by ingestion of the bacteria or a tissue of the transgenic plant; thereafter, the growth of Chilo suppressalis is inhibited and/or Chilo suppressalis dies, so as to achieve controlling the damage of Chilo suppressalis to the plant.

4. A method according to claim 3, wherein the tissues of the transgenic plant are roots, 15 leaves, stems, fruits, tassels, ears, anthers or filaments.

5. A method according to claim 3 or 4, wherein the plant is selected from the group consisting of rice, sugar cane, wild rice, corn, sorghum, soybean, rape, wheat, millet and barnyard.

6. A method according to any one of claims 3 to 5, wherein prior to contacting, the method 20 includes a step of planting a seedling containing a polynucleotide encoding Vip3A protein.

7. A method according to any one of claims 3 to 6, wherein the nucleotide sequence encoding Vip3A protein comprises a nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:4.

8. A method according to any one of claims 3 to 7, wherein the plant also contains at least a 25 second nucleotide sequence which is different from the nucleotide sequence encoding

Vip3A protein.

9. A method according to claim 8, wherein the second nucleotide sequence encodes a Cry-type insecticidal protein, a Vip-type insecticidal protein, a protease inhibitor, lectin, α-amylase, peroxidase or a dsRNA inhibiting an important gene of target insect or pest.

30 10. A method according to claim 9, wherein the second nucleotide sequence encodes

CrylAb protein.

11. A method according to claim 10, wherein the amino acid sequence of CrylAb protein comprises an amino acid sequence shown by SEQ ID NO:5.

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12. A method according to claim 11, wherein the second nucleotide comprises a nucleotide sequence shown by SEQ ID NO:6.

13. Use of a Vip3A protein comprising an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 to control Chilo suppressalis.

5 14. Use of a plant cell, a plant tissue, a plant or a bacterium which is transformed with a

Vip3A gene encoding Vip3A protein comprising an amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:3 to control Chilo suppressalis.

15. A method for producing a plant that controls Chilo suppressalis, comprising the step of introducing a polynucleotide sequence encoding Vip3A protein comprising an amino acid

10 sequence shown in SEQ ID NO:1 or SEQ ID NO:3 into the genome of a plant, wherein the expression of said polynucleotide in the plant controls Chilo suppressalis.

16. A method for producing a propagule that controls Chilo suppressalis, wherein the method includes a step of hybridizing a first plant produced by a method according to claim 15 with a second plant, and/or removing the tissue having the reproductive capacity from

15 the plant produced by a method according to claim 15 and culturing same to produce a propagule containing a polynucleotide sequence encoding Vip3A protein.

17. A method for cultivating a plant that controls Chilo suppressalis, wherein the method includes the following steps:

i) planting at least one propagule whose genome contains a polynucleotide sequence 20 encoding Vip3A protein comprising an amino acid sequence shown in SEQ ID NO:1 or

SEQ ID NO:3;

making the propagule grow into a plant;

making the plant grow under the condition artificial inoculation and/or natural occurrence of Chilo suppressalis, and harvesting the plant with weakened plant damage and/or

25 increased plant yield compared to other plant without the polynucleotide sequence encoding Vip3A protein.

18. A propagule, a plant cell, a plant tissue or a plant that controls Chilo suppressalis, wherein the propagule, the plant cell, the plant tissue or the plant contains the polynucleotide encoding Vip3A protein that comprises an amino acid sequence shown in

30 SEQ ID NO:1 or SEQ ID NO:3, wherein the expression of said polynucleotide when used in the plant controls Chilo suppressalis.

19. A propagule, a plant cell, a plant tissue or a plant according to claim 18, wherein the nucleotide sequence encoding Vip3A protein comprises a nucleotide sequence shown in SEQ ID NO:2 or SEQ ID NO:4.

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20. A propagule, a plant cell, a plant tissue or a plant according to claim 18 or 19, wherein the propagule, the plant cell, the plant tissue or the plant at least contains a second nucleotide sequence which is different from the nucleotide sequence encoding the Vip3A protein.