CN106749566B

CN106749566B - Insecticidal protein combinations and methods of managing insect resistance

Info

Publication number: CN106749566B
Application number: CN201611039248.4A
Authority: CN
Inventors: 陶青; 杨旭; 李建勇
Original assignee: Beijing Dabeinong Technology Group Co Ltd; Beijing Dabeinong Biotechnology Co Ltd
Current assignee: Beijing Dabeinong Biotechnology Co Ltd
Priority date: 2016-11-21
Filing date: 2016-11-21
Publication date: 2020-05-05
Anticipated expiration: 2036-11-21
Also published as: WO2018090714A1; CN106749566A

Abstract

The present invention relates to an insecticidal protein combination and a method for managing insect resistance thereof, which comprises contacting Asian corn borer with at least Cry2Ab protein and Cry1Ac protein. According to the invention, resistance of the Asiatic corn borers is effectively delayed or prevented by utilizing two insecticidal proteins Cry1Ac and Cry2Ab, so that the control or prevention of the Asiatic corn borers is realized, and plants are protected to a greater extent and the yield is stabilized.

Description

Insecticidal protein combinations and methods of managing insect resistance

Technical Field

The invention relates to an insecticidal protein combination and a method for managing insect resistance, in particular to a method for managing Asian corn borer resistance by using Cry1Ac protein and Cry2Ab protein in combination.

Background

Insect pests inflict huge economic losses on global crop production, and farmers face the threat of yield loss due to insect pests every year. Asiatic corn borer (Ostriniafnacalis) is commonly named as borer, belongs to the family of Lepidoptera borer, is a main pest in corn production in China, and the insect eats corn leaves and moths into corn main stems or fruit ears, so that photosynthesis is reduced, nutrient transportation is influenced, various secondary diseases are generated, and the yield and the quality of the corn are reduced. In recent years, along with the change of climate conditions, the change of farming systems, the increase of corn planting density and the improvement of water fertilizing conditions, the harm of the Asiatic corn borers is increasingly aggravated.

The ability to produce insect-resistant plants by transforming Bt (Bacillus thuringiensis) insecticidal protein genes revolutionized modern agriculture and increased the importance and value of insecticidal proteins and their genes. Several Bt proteins have been used in transgenic plants to confer insect resistance, including the Cry1Ab protein, the Cry1Ac protein, the Cry1F protein, the Cry2Ab protein, the Cry3Bb protein, and the Vip3A protein, among others. However, with the widespread use of transgenic crops, insects will develop resistance to Bt proteins expressed in transgenic plants under continuous selection pressure, and such resistance, once developed and not effectively controlled, will undoubtedly limit the commercial value of transgenic crop varieties containing Bt proteins. Only by implementing a reasonable resistance management strategy can the modern technical result be durably utilized.

In order to reduce the generation of insect resistance, the following resistance management strategies are mainly adopted in production:

1) and (5) arranging shelters in the field. Refuges were set up to maintain a proportion of non-resistant alleles to delay the production of insects that tolerate high doses of insect-resistant protein, but this strategy would make the farmer cumbersome to operate and reduce overall production to some extent.

2) The strategy is less sustainable by increasing the dosage of a single insect-resistant protein, and insects will have higher resistance to the single protein under continuous selection pressure.

3) Different insect-resistant proteins are used alternately or together. Since Bt protein specifically binds to a receptor of a sensitive insect, the overlapping use of insect-resistant proteins requires high uncertainty in evaluating whether insects have cross-resistance to different insect-resistant proteins, i.e., whether the insects share or compete for binding sites, and thus, no report on whether Cry2Ab protein has cross-resistance to Asian corn borer and Cry1Ac protein exists so far.

Disclosure of Invention

The invention aims to provide an insecticidal protein combination and a method for managing insect resistance, and provides a method for controlling resistant Asian corn borers through the combination of Cry2Ab protein and Cry1Ac protein for the first time, and effectively delays the resistance of the Asian corn borers to a single protein.

To achieve the above objects, the present invention provides a method for managing insect resistance, which comprises contacting Asian corn borer with at least Cry2Ab protein and Cry1Ac protein.

Further, the Cry2Ab protein and Cry1Ac protein are present in a bacterium or transgenic plant producing at least the Cry2Ab protein and Cry1Ac protein, and the Asian corn borer is contacted with at least the Cry2Ab protein and Cry1Ac protein by ingestion of a tissue of the bacterium or the transgenic plant, and growth of the Asian corn borer is inhibited and/or caused to die after contact, to achieve management of resistance to the Asian corn borer.

Still further, Asian corn borers resistant to a Cry1Ac protein are contacted with at least said Cry2Ab protein, said Cry2Ab protein is present in a bacterium or a transgenic plant that produces at least said Cry2Ab protein, said Asian corn borers resistant to a Cry1Ac protein are contacted with at least said Cry2Ab protein by ingestion of a tissue of said bacterium or said transgenic plant, and growth of said Asian corn borers resistant to a Cry1Ac protein is inhibited and/or caused to die upon contact to achieve management of resistance of said Asian corn borers resistant to a Cry1Ac protein.

Still further, an asian corn borer resistant to a Cry2Ab protein is contacted with at least said Cry1Ac protein, said Cry1Ac protein is present in a bacterium or a transgenic plant that produces at least said Cry1Ac protein, said asian corn borer resistant to a Cry2Ab protein is contacted with at least said Cry1Ac protein by ingestion of a tissue of said bacterium or said transgenic plant, and upon contact said asian corn borer resistant to a Cry2Ab protein growth is inhibited and/or caused to die to achieve management of said resistance to an asian corn borer resistant to a Cry2Ab protein.

In the above technical scheme, the transgenic plant is in any growth period.

Further, the tissue of the transgenic plant is root, leaf, stem, fruit, tassel, ear, anther or filament.

Still further, the control of asian corn borer dangerous plants is not altered by changes in planting location and/or planting time.

Alternatively, the plant is maize, wheat, sorghum, millet, rice or soybean.

In the technical scheme, the amino acid sequence of the Cry2Ab protein has the amino acid sequence shown as SEQ ID NO. 1.

Further, the amino acid sequence of the Cry2Ab protein has an amino acid sequence shown as SEQ ID NO. 1.

Preferably, the amino acid sequence of the Cry1Ac protein has an amino acid sequence shown as SEQ ID NO. 2.

Further, the nucleotide sequence of the Cry1Ac protein has a nucleotide sequence shown as SEQ ID NO. 4.

To achieve the above objects, the present invention also provides a method for controlling asian corn borer, which comprises contacting the asian corn borer with at least Cry2Ab protein and Cry1Ac protein, thereby achieving control of the asian corn borer.

Further, the Cry2Ab protein and the Cry1Ac protein are present in a bacterium or a transgenic plant which produces at least the Cry2Ab protein and the Cry1Ac protein, the Asian corn borer is contacted with at least the Cry2Ab protein and the Cry1Ac protein by ingestion of a tissue of the bacterium or the transgenic plant, and growth of the Asian corn borer is inhibited and/or caused to die after the contact, so as to realize control of the Asian corn borer harmful plants.

Still further, Asian corn borers resistant to a Cry1Ac protein are contacted with at least said Cry2Ab protein, said Cry2Ab protein is present in a bacterium or a transgenic plant that produces at least said Cry2Ab protein, said Asian corn borers resistant to a Cry1Ac protein are contacted with at least said Cry2Ab protein by ingestion of a tissue of said bacterium or said transgenic plant, and growth of said Asian corn borers resistant to a Cry1Ac protein is inhibited and/or caused to die after contact to effect control of said Asian corn borers resistant to a Cry1Ac protein compromising the plant.

Still further, an asian corn borer resistant to a Cry2Ab protein is contacted with at least said Cry1Ac protein, said Cry1Ac protein is present in a bacterium or a transgenic plant that produces at least said Cry1Ac protein, said asian corn borer resistant to a Cry2Ab protein is contacted with at least said Cry1Ac protein by ingestion of a tissue of said bacterium or said transgenic plant, and growth of said asian corn borer resistant to a Cry2Ab protein is inhibited and/or caused to die upon contact to effect control of said asian corn borer resistant to a Cry2Ab protein compromised plant.

In the above technical scheme, the transgenic plant is in any growth period.

Preferably, the control of asian corn borer dangerous plants is not altered by changes in planting location and/or planting time.

Alternatively, the plant is maize, wheat, sorghum, millet, rice or soybean.

Further, the nucleotide sequence of the Cry2Ab protein has a nucleotide sequence shown as SEQ ID NO. 3.

In order to achieve the aim, the invention provides the application of the Cry2Ab protein and the Cry1Ac protein in combination so as to prevent or delay the resistance of Asian corn borer populations to the Cry1Ac protein or the Cry2Ab protein.

In order to achieve the aim, the invention also provides the application of the Cry2Ab protein and the Cry1Ac protein which are used in combination to control the Asian corn borer population which generates resistance to the Cry1Ac protein or the Cry2Ab protein.

"contacting" as used herein means that the insect and/or pest touches, dwells and/or ingests a plant, plant organ, plant tissue or plant cell which either expresses an insecticidal protein in vivo or has an insecticidal protein on the surface and/or has a microorganism producing an insecticidal protein.

The invention relates to the 'control' and/or 'control' which means that Asian corn borer is contacted with at least Cry2Ab protein and Cry1Ac protein, after the contact, the growth of the Asian corn borer is inhibited and/or the Asian corn borer dies. Further, the Asian corn borer pests are contacted with at least Cry2Ab protein and Cry1Ac protein by feeding plant tissues, and after the contact, all or part of the Asian corn borers are inhibited from growing and/or die. Inhibition refers to sublethal, i.e., not yet lethal, but capable of causing some effect in growth, development, behavior, physiology, biochemistry and tissue, such as slow and/or stopped growth. At the same time, the plant should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product. In addition, the plants and/or plant seeds for controlling Asian corn borer pests, which contain the polynucleotide sequences encoding the Cry2Ab protein and the Cry1Ac protein, have reduced plant damage, including but not limited to improved stalk resistance, and/or increased kernel weight, and/or yield increase, and the like, compared to non-transgenic wild-type plants, under conditions in which the Asian corn borer pests and/or the Asian corn borer pests are naturally harmed by artificial inoculation. The Cry2Ab protein and the Cry1Ac protein can independently exist on the control and/or prevention effects of the Asiatic corn borer, and are not weakened and/or disappeared by the existence of other substances which can control and/or prevent the Asiatic corn borer pests. In particular, any tissue of the transgenic plant (containing the polynucleotide sequence encoding the Cry2Ab protein and the Cry1Ac protein) that is present and/or produced, simultaneously and/or asynchronously, both the Cry2Ab protein and the Cry1Ac protein and/or another substance that can control asian corn borer pests, then the presence of said another substance does not affect, nor does it result in said "controlling" and/or "controlling" action of the Cry2Ab protein and the Cry1Ac protein on asian corn borer, being completely and/or partially effected by said another substance, regardless of the Cry2Ab protein and the Cry1Ac protein. Generally, the feeding process of the Asian corn borer pests to plant tissues in the field is short and difficult to observe visually, so that dead Asian corn borer pests exist in any tissues of transgenic plants (containing polynucleotide sequences encoding the Cry2Ab protein and the Cry1Ac protein) under the condition of the natural occurrence of harm of the artificially inoculated Asian corn borer pests and/or the Asian corn borer pests, and/or the Asian corn borer pests are stopped and growth of the killed Asian corn borer pests is inhibited, and/or the transgenic wild-type plants have reduced plant damage, namely, the method and/or the application of the invention are realized, namely, the method and/or the application of controlling the Asian corn borer pests are realized by the contact of the Asian corn borer pests with at least the Cry2Ab protein and the Cry1Ac protein.

The term "plant" includes whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps (plant granules), and plant cells intact in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, and the like.

The genome of a plant, plant tissue or plant cell as defined in the present invention refers to any genetic material within a plant, plant tissue or plant cell and includes the nuclear and plastid and mitochondrial genomes.

"recombinant" as referred to herein refers to a form of DNA and/or protein and/or organism that is not normally found in nature and is therefore produced by human intervention. Such manual intervention may result in recombinant DNA molecules and/or recombinant plants. Such "recombinant DNA molecules" are obtained by artificially combining two otherwise isolated sequence segments, for example, by chemical synthesis or by manipulating an isolated nucleic acid segment by genetic engineering techniques. Techniques for performing nucleic acid manipulations are well known.

The term "protoxin" or "toxin" or "pesticidal toxin" of the present invention refers to the initial translation product of the full-length gene encoding the pesticidal protein prior to any break in the midgut. The term "toxin" or "least toxic fragment" according to the present invention is to be understood as a part of an insecticidal protein, such as the Cry2Ab or Cry1Ac protein, which can be obtained by trypsin digestion or by hydrolysis in the midgut fluid of (target insects, such as asian corn borer) and still have insecticidal activity. Typically on SDS-PAGE gels, the Cry1 toxin has a molecular weight of about 60-65kD and the Cry2A toxin has a molecular weight of about 50-58 kD.

In the present invention, the Cry2Ab protein and the Cry1Ac protein are expressed in a transgenic plant. Co-expression of more than one insecticidal toxin in the same transgenic plant can be achieved by genetic engineering the plant to contain and express the desired genes. Alternatively, one plant (the 1 st parent) may be genetically engineered to express a Cry2Ab protein and a second plant (the 2 nd parent) may be genetically engineered to express a Cry1Ac protein. Progeny plants expressing all the genes introduced into the 1 st and 2 nd parents are obtained by crossing the 1 st and 2 nd parents.

In the present invention, transgenic plants that produce the Cry1Ac protein include, but are not limited to, the TT51 transgenic rice event and/or plant material comprising the TT51 transgenic rice event (as described in CN100582223C and CN 101302520B), the 223F-S21 transgenic rice line and/or plant material comprising the 223F-S21 transgenic rice line (as described in CN 103773759A), the Mon15985 transgenic cotton event and/or plant material comprising the Mon15985 transgenic cotton event (as described in CN 101413028B), the Mon transgenic cotton event and/or plant material comprising the Mon531 transgenic cotton event (as described in USDA hiaps non-regulated state application 00-342-01 p), the COT67B transgenic cotton event and/or plant material comprising the COT67B transgenic cotton event (as described in USDA hiaps non-regulated state application 07-108-01 p) or the 3006-23 transgenic cotton event and/or plant material comprising the COT 36531 transgenic cotton event 3006-210-23 transgenic Cotton event plant material (as described in USDA APHIS unregulated state application 03-036-02 p).

In the present invention, transgenic plants that produce the Cry2Ab protein include, but are not limited to, a Mon89034 transgenic corn event and/or plant material comprising a Mon89034 transgenic corn event (as described in CN 101495635A), a Mon87751 transgenic soybean event and/or plant material comprising a Mon87751 transgenic soybean event (as described in USDAAPHIS non-regulatory state application 13-337-01 p), or a Mon15985 transgenic cotton event and/or plant material comprising a Mon15985 transgenic cotton event (as described in CN 101413028B).

As is well known to those skilled in the art, DNA typically exists in a double stranded form. In this arrangement, one strand is complementary to the other strand, and vice versa. Other complementary strands of DNA are produced as the DNA replicates in plants. Thus, the present invention includes the use of the polynucleotides and their complementary strands exemplified in the sequence listing. The "coding strand" as commonly used in the art refers to the strand to which the antisense strand is joined. To express a protein in vivo, one strand of DNA is typically transcribed into the complementary strand of an mRNA, which serves as a template for translation of the protein. mRNA is actually transcribed from the "antisense" strand of DNA. The "sense" or "coding" strand has a series of codons (a codon is three nucleotides, three of which at a time can yield a particular amino acid) that can be read as an Open Reading Frame (ORF) to form a protein or peptide of interest. The present invention also includes RNAs that are functional equivalent to the exemplified DNA.

Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the pesticidal gene of the present invention. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. In the present invention, two nucleic acid molecules can be said to be capable of specifically hybridizing to each other if they can form an antiparallel double-stranded nucleic acid structure. Two nucleic acid molecules are said to be "complements" of one another if they exhibit complete complementarity. In the present invention, two nucleic acid molecules are said to exhibit "perfect complementarity" when each nucleotide of the two nucleic acid molecules is complementary to the corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability to allow them to anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to have "complementarity" if they are capable of hybridizing to each other with sufficient stability to allow them to anneal and bind to each other under conventional "highly stringent" conditions. Deviations from perfect complementarity may be tolerated as long as such deviations do not completely prevent the formation of a double-stranded structure by the two molecules. In order to allow a nucleic acid molecule to act as a primer or probe, it is only necessary to ensure sufficient complementarity in sequence to allow the formation of a stable double-stranded structure in the particular solvent and salt concentrations employed.

In the present invention, a substantially homologous sequence is a nucleic acid molecule that specifically hybridizes under highly stringent conditions to the complementary strand of a compatible nucleic acid molecule. Suitable stringency conditions for promoting DNA hybridization include, for example, treatment with 6.0 XSSC/sodium citrate (SSC) at about 45 ℃ followed by a wash with 2.0 XSSC at 50 ℃, as is well known to those skilled in the art. For example, the salt concentration in the washing step can be selected from the group consisting of about 2.0 XSSC for low stringency conditions, 50 ℃ to about 0.2 XSSC for high stringency conditions, 50 ℃. In addition, the temperature conditions in the washing step can be raised from about 22 ℃ at room temperature for low stringency conditions to about 65 ℃ for high stringency conditions. Both the temperature conditions and the salt concentration may be varied, or one may be held constant while the other is varied. Preferably, the stringent conditions of the present invention may be those which specifically hybridize to SEQ ID NO:3 and SEQ ID NO:4 in a 6 XSSC, 0.5% SDS solution at 65 ℃ and then wash the membrane 1 time each with 2 XSSC, 0.1% SDS, and 1 XSSC, 0.1% SDS.

Thus, sequences having anti-insect activity and which hybridize under stringent conditions to SEQ ID NO. 3 or SEQ ID NO. 4 of the present invention are included in the present invention. These sequences are at least about 40% -50% homologous, about 60%, 65%, or 70% homologous, and even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence homology to the sequences of the present invention.

The genes and proteins described in the present invention include not only the specific exemplified sequences, but also portions and/or fragments (including internal and/or terminal deletions compared to the full-length protein), variants, mutants, substitutions (proteins with substituted amino acids), chimeras and fusion proteins that preserve the pesticidal activity characteristics of the specific exemplified proteins. The "variant" or "variation" refers to a nucleotide sequence that encodes the same protein or encodes an equivalent protein with pesticidal activity. The "equivalent protein" refers to a protein having the same or substantially the same biological activity against a lepidopteran insect pest as the protein of claim.

"fragment" or "truncation" of a DNA molecule or protein sequence as described herein refers to a portion of the original DNA or protein sequence (nucleotide or amino acid) or an artificially modified form thereof (e.g., a sequence suitable for plant expression) that may vary in length but is long enough to ensure that the (encoded) protein is an insect toxin.

Modification of genes and easy construction of gene variants can be achieved using standard techniques. For example, techniques for making point mutations are well known in the art. Another example is U.S. patent No. 5605793, which describes methods for generating other molecular diversity using DNA reassembly after random fragmentation. Fragments of the full-length gene can be made using commercial endonucleases, and exonucleases can be used following standard procedures. For example, nucleotides can be systematically excised from the ends of these genes using enzymes such as Bal31 or site-directed mutagenesis. A variety of restriction enzymes can also be used to obtain a gene encoding an active fragment. Active fragments of these toxins can be obtained directly using proteases.

The present invention may derive equivalent proteins and/or genes encoding these equivalent proteins from b.t. isolates and/or DNA libraries. There are various methods for obtaining the pesticidal proteins of the present invention. For example, antibodies to the pesticidal proteins disclosed and claimed herein can be used to identify and isolate other proteins from a mixture of proteins. In particular, antibodies may be caused by the most constant and different protein portions of the protein than other Bt proteins. These antibodies can then be used to specifically identify the equivalent proteins with characteristic activities by immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) or western blot methods. Antibodies to the proteins disclosed in the present invention or equivalent proteins or fragments of such proteins can be readily prepared using standard procedures in the art. The genes encoding these proteins can then be obtained from the microorganism.

Due to the redundancy of the genetic code, a plurality of different DNA sequences may encode the same amino acid sequence. It is well within the skill of the art to generate such alternative DNA sequences encoding the same or substantially the same protein. These different DNA sequences are included in the scope of the present invention. The "substantially identical" sequence refers to a sequence having amino acid substitutions, deletions, additions or insertions which do not substantially affect pesticidal activity, and also includes fragments which retain pesticidal activity.

The substitution, deletion or addition of the amino acid sequence in the present invention is a conventional technique in the art, and it is preferable that such amino acid change is: small changes in properties, i.e., conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of about 1-30 amino acids; a small amino-or carboxy-terminal extension, e.g., one methionine residue to the amino terminus; small linker peptides, for example, about 20-25 residues in length.

Examples of conservative substitutions are those that occur within the following amino acid groups: basic amino acids (e.g., arginine, lysine, and histidine), acidic amino acids (e.g., glutamic acid and aspartic acid), polar amino acids (e.g., glutamine, asparagine), hydrophobic amino acids (e.g., leucine, isoleucine, and valine), aromatic amino acids (e.g., phenylalanine, tryptophan, and tyrosine), and small molecule amino acids (e.g., glycine, alanine, serine, threonine, and methionine). Those amino acid substitutions which do not normally alter a particular activity are well known in the art and have been described, for example, by N.Neurath and R.L.Hill in Protein, 1979, New York Academic Press. The most common exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thu/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 vice versa.

It will be apparent to those skilled in the art that such substitutions may occur outside the region which plays an important role in the function of the molecule and still result in an active polypeptide. For polypeptides of the invention whose activity is essential and therefore the choice of unsubstituted amino acid residues can be identified according to methods known in 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 involves introducing mutations at each positively charged residue in the molecule and testing the resulting mutant molecules for anti-insect activity to determine amino acid residues important for the activity of the molecule. The substrate-enzyme interaction site can also be determined by analysis of its three-dimensional structure, which can be determined by techniques such as nuclear magnetic resonance analysis, crystallography, or photoaffinity labeling (see, e.g., de Vos et al, 1992, Science 255: 306-.

Thus, amino acid sequences having a certain homology with the amino acid sequences shown in SEQ ID NO. 1 and/or SEQ ID NO. 2 are also included in the present invention. These sequences typically have a similarity/identity of greater than 78%, preferably greater than 85%, more preferably greater than 90%, even more preferably greater than 95%, and may be greater than 99% to the sequences of the present invention. Preferred polynucleotides and proteins of the invention may also be defined according to more specific identity and/or similarity ranges. For example 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity and/or similarity to a sequence exemplified herein.

The term "cross-resistance" as used herein refers to the phenomenon of a strain of an insect that is resistant to an agent or class of agents other than the selected agent that has never been used due to the same resistance mechanism or a similar mechanism of action or a similar chemical structure. In particular, the term "cross-resistance" as used herein refers to the phenomenon of resistance to an insecticidal protein or class of insecticidal proteins other than the selected insecticidal protein that has never been used. The proteins selected for insect resistance management need to exert their pesticidal effects independently so that resistance to one protein does not confer resistance to a second protein (i.e., no cross-resistance or lower cross-resistance to a second protein). For example, a population of insects resistant to "protein a" is sensitive to "protein B", one can conclude that protein a and protein B are not cross-resistant and that their combination is effective in delaying resistance to a single protein a.

The term "resistance ratio" refers to the fold increase of the same insecticidal protein in the Bt resistant population of insects relative to the LC50 value of the Bt sensitive population, i.e. the resistance ratio is Bt resistant strain LC50 value/Bt sensitive strain LC50 value.

As used herein, a population of insect species "resistant", "already resistant" or "made resistant" to an insecticidal protein and/or a plant expressing an insecticidal protein that previously controlled or killed said insect population means that a repetitive and significant yield loss is detected in the plant resulting from a diminished controlling effect of the insect population and/or resulting from a level of yield loss in the plant as compared to the controlling effect in the same insect species when the insecticidal protein was first introduced and/or the plant and/or resulting from the same insect species.

Asiatic corn borer (Ostriniafnacalis) and Heliothis armigera (Helicoverpa armigera Hubner) although belong to the same Lepidoptera, the Asiatic corn borer and Helicoverpa armigera are two biologically distinct and distinct species, with at least the following major differences: 1) asian corn borer belongs to the family of borer moth and cotton bollworm belongs to the family of noctuidae. 2) The food property is different. The Asiatic corn borers most commonly damage corns, can damage various parts on the ground of corn plants, including leaves, tassels, stalks, ear stalks and cob, and have different degrees of damage to the corns sowed in spring, summer and autumn in various places, particularly the corn sowed in summer is the heaviest; bollworms are important boring pests that harm the bud and boll stage of cotton, and mainly eat buds, flowers and bolls, and also eat tender leaves. 3) The distribution areas are different. The Asiatic corn borers are distributed in the east and southwest regions of China where main corns and broomcorn are produced; cotton bollworms are widely distributed in China and all over the world, cotton areas in China and vegetable planting areas occur, and the cotton areas in the yellow river basin and the Yangtze river basin are seriously damaged; in recent years, the cotton area in Xinjiang has also occurred. 4) The morphological features are different. The Asiatic corn borer and the cotton bollworm have great difference in the morphological characteristics of eggs, larvae, pupae and imagoes. 5) Growth habit and occurrence law are different. The generation passage number of the corn borer is obviously different with latitude: in China, the north latitude 45 degrees is in the north 1 generation, 45 degrees to 40 degrees 2 generation, 40 degrees to 30 degrees 3 generation, 30 degrees to 25 degrees 4 generation, 25 degrees to 20 degrees 5 to 6 generation. The higher the altitude, the fewer generations occur; the pupa pupae grow in the next 4-5 months and grow after about 10 days, wherein the generation is 2-4 generations in Sichuan province in one year, the temperature is high, the altitude is low, and the generation number is more, and the mature larvae generally live through the winter in maize stalks, cobs or sorghum and sunflower stalks. Adults move at night, have strong flying power and phototaxis, have a service life of 5-10 days, like to lay eggs on two sides of a midrib on the back of a corn leaf which is over 50 cm away from the ground and has more vigorous growth, one female moth can lay eggs for 350 plus 700 grains, and the egg period is 3-5 days; the corn borers are suitable for developing under the conditions of high temperature and high humidity, the temperature is higher in winter, the parasitic amount of natural enemies is small, the propagation of the corn borers is facilitated, and the harm is serious; in the egg period, the corn leaves are curled due to drought, egg masses are easy to fall off from the back of the leaves to die, and the harm is light. The generation number of cotton bollworms varies from year to year and 4 generations of cotton bollworms occur in Laizhou city, Shandong province, the grown larvae successively enter the tree and enter the soil in late September, and pupate in the soil near nursery stocks or 5-10cm deep below weeds for overwintering; the eclosion begins when the temperature rises back to more than 15 ℃ in the beginning of spring, the full eclosion period is from late 4 months to late 5 months, adults appear in the first middle and late 6 months, the second generation appears in the middle and late 7 months, cotton bollworms appear in the third generation from late 8 months to late 9 months to late 10 months, the adults have phototaxis, after eclosion, eggs are flashed to lay at night, the eggs are scattered, relatively scattered, 500 plus eggs can be laid by one female moth for one life, the maximum can reach 2700 eggs, the eggs are mostly laid on the back of leaves, and also can be laid on other plants such as the front, the top core, the petioles, the tender stems or agricultural crops and weeds; after the larvae are hatched, the larvae have a feeding egg shell habit, the larvae which are hatched initially have a cluster food limit habit, the two, three and three five heads are arranged on the front side or the back side of the blade, the heads are arranged towards the leaf margin and feed inwards from the leaf margin, and as a result, the blade is eaten and only main veins and leaf stalks are left or withers in a net shape, so that dry leaves are caused; 1-2 instar larva moves down along the stem to the top bud of the ginkgo seedling and eats from one side or eats into a twig along the top bud, so that the top end or top cluster leaves die, and the damage is very serious; the food intake of the larvae before 3 th instar is less and more concentrated, the larvae gradually disperse along with the growth of the larvae, the food intake of the larvae enters 4 th instar and is greatly increased, the larvae can eat smooth leaves, and only petioles are left; the larvae are most harmful in 7-8 months; cotton bollworm has the habit of transferring harm, and one larva can harm multiple seedlings; the larvae of all ages have the habit of eating the old skin after sloughing and leaving head shells, so that certain difficulty is caused in identifying the ages of the larvae, and the larvae are not regular in age; the optimum temperature for the cotton bollworm is 25-28 ℃ and the relative humidity is 70-90%; the second generation and the third generation are the most serious, the population density of the pests in the serious field reaches 98 heads/shutter, the pest rate is 60-70%, the individual field reaches 100%, the damaged leaves reach more than 1/3, the yield of the leaves is influenced by 20%, the quality is reduced by at least 1 grade, and the seedling growth quantity is greatly influenced.

In summary, although the Asian corn borers and the cotton bollworms belong to the same Lepidoptera, the Asian corn borers and the cotton bollworms have various differences only in morphological characteristics and habits, and are far away from each other in genetic relationship, so that the Asian corn borers and the cotton bollworms cannot mate to generate offspring. Thus, the receptors on the cell membrane surface of the intestinal epithelium that bind Bt toxin are also different between the two.

The resistance mechanism of insects to Bt proteins is not single, Heckel (1994) et al analyzed the potential mechanism of insect resistance to Bt proteins, and it is thought that the development of resistance is mainly related to the following factors: 1) solubility of Bt insecticidal proteins: protoxin insolubility or solubility reduction; 2) proteolytic properties of Bt protoxin: insufficient or excessive hydrolysis; 3) binding of Bt proteins to receptors on cell membranes: the combination of the Bt protein and the receptor is blocked due to competitive inhibition, and the modification effect of the primary structure or the secondary structure of the receptor is changed, so that the combination sites of the Bt protein and the receptor are reduced; 4) formation of cavities on the cell membrane: the formation of voids is hindered or blocked; 5) repairing function of midgut epithelium; 6) the mechanism of action, etc. Wherein the alteration of the binding capacity of the toxin to receptors on the cell membrane is the main resistance mechanism.

When two or more different Bt proteins share a binding site in insects, they are not able to provide a good combination for insect resistance management purposes. In practice, there is a high uncertainty as to which resistance management strategy to adopt for different insects. Two Bt proteins with large differences in amino acid sequence may also bind to a common binding site with high affinity in certain insect species, for example Cry1Ab and Cry1F proteins in Plutella xylostella (Plutella xylostella). Furthermore, it has been found that two proteins that do not have a shared binding site in one insect species can share a binding site in another insect species, for example Fiuza et al (1996) found that Cry1Ac and Cry1Ba proteins share a binding site in Chilo supressalis (Chilo supressalis), whereas Ballester et al (1999) found that the above two proteins bind different binding sites in cabbage moth.

In the invention, the Cry2Ab protein can have an amino acid sequence shown as SEQ ID NO. 1 in a sequence table; the Cry1Ac protein can have an amino acid sequence shown as SEQ ID NO. 2 in a sequence table. In addition to comprising the coding regions for the Cry2Ab protein and the Cry1Ac protein, which may also comprise other elements, such as coding regions encoding transit peptides or encoding selectable marker proteins, the nucleotide sequences for the Cry2Ab protein and the Cry1Ac protein provided herein may be formed into constructs by conventional means.

In addition, the construct comprising proteins encoding the Cry2Ab protein and Cry1Ac protein of the invention can also be expressed in plants together with at least one protein encoding a herbicide resistance gene, including, but not limited to, a phosphinothricin resistance gene (e.g., bar gene, pat gene), a bendiocide resistance gene (e.g., pmph gene), a glyphosate resistance gene (e.g., EPSPS gene), a bromoxynil (broloxynil) resistance gene, a sulfonylurea resistance gene, a resistance gene to the herbicide dalapon, a resistance gene to cyanamide, or a resistance gene to a glutamine synthetase inhibitor (e.g., PPT), thereby obtaining transgenic plants having both high pesticidal activity and herbicide resistance.

The regulatory sequences of the present invention include, but are not limited to, promoters, transit peptides, terminators, enhancers, leaders, introns, and other regulatory sequences operably linked to the Cry2Ab protein or the Cry1Ac protein.

The promoter is a promoter capable of being expressed in a plant, and the promoter capable of being expressed in the plant is a promoter which ensures that a coding sequence connected with the promoter is expressed in a plant cell. The promoter expressible in plants may be a constitutive promoter. Examples of promoters that direct constitutive expression in plants include, but are not limited to, 35S promoter derived from cauliflower mosaic virus, maize Ubi promoter, promoter of rice GOS2 gene, and the like. Alternatively, the plant expressible promoter may be a tissue specific promoter, i.e. a promoter that directs expression of the coding sequence at a higher level in some tissues of the plant, e.g. in green tissues, than in other tissues of the plant (as can be determined by conventional RNA assays), e.g. the PEP carboxylase promoter. Alternatively, the promoter expressible in a plant may be a wound-inducible promoter. A wound-inducible promoter or a promoter that directs a wound-induced expression pattern means that when a plant is subjected to mechanical or insect feeding induced wounds, the expression of the coding sequence under the control of the promoter is significantly increased compared to under normal growth conditions. Examples of wound-inducible promoters include, but are not limited to, promoters of potato and tomato protease-inhibitory genes (pin I and pin II) and maize protease-inhibitory gene (MPI).

The transit peptide (also known as a secretion signal sequence or targeting sequence) is intended to direct the transgene product to a specific organelle or cellular compartment, and for the receptor protein, the transit peptide may be heterologous, e.g., targeting the chloroplast using a chloroplast transit peptide sequence, or targeting the endoplasmic reticulum using a 'KDEL' retention sequence, or targeting the vacuole using the CTPP of the barley lectin gene.

The leader sequence includes, but is not limited to, a small RNA virus leader sequence, such as an EMCV leader sequence (encephalomyocarditis virus 5' non-coding region); potyvirus leaders, such as the MDMV (maize dwarf mosaic virus) leader; human immunoglobulin heavy chain binding protein (BiP); untranslated leader sequence of envelope protein mRNA of alfalfa mosaic virus (AMVRNA 4); tobacco Mosaic Virus (TMV) leader sequence.

Such enhancers include, but are not limited to, cauliflower mosaic virus (CaMV) enhancer, Figwort Mosaic Virus (FMV) enhancer, carnation weathering Circovirus (CERV) enhancer, cassava vein mosaic virus (CsVMV) enhancer, Mirabilis Mosaic Virus (MMV) enhancer, midnight fragrant tree yellowing leaf curl virus (CmYLCV) enhancer, multan cotton leaf curl virus (CLCuMV), dayflower yellow mottle virus (CoYMV), and peanut chlorosis streak mosaic virus (PCLSV) enhancer.

For monocot applications, the intron includes, but is not limited to, the maize hsp70 intron, the maize ubiquitin intron, Adh intron 1, the sucrose synthase intron, or the rice Act1 intron. For dicot applications, the introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "superubiquitin" intron.

The terminator may be a suitable polyadenylation signal sequence that functions in plants, including, but not limited to, polyadenylation signal sequence derived from the Agrobacterium tumefaciens nopaline synthase (NOS) gene, polyadenylation signal sequence derived from the protease inhibitor II (PIN II) gene, polyadenylation signal sequence derived from the pea ssRUBISCO E9 gene, and polyadenylation signal sequence derived from the α -tubulin (α -tubulin) gene.

As used herein, "operably linked" refers to the linkage of nucleic acid sequences such that one provides the functionality required of the linked sequence. In the present invention, the "operative linkage" may be a linkage of a promoter to a sequence of interest such that transcription of the sequence of interest is controlled and regulated by the promoter. "operably linked" when the sequence of interest encodes a protein and expression of the protein is desired indicates that: the promoter is linked to the sequence in such a way that the resulting transcript is translated efficiently. If the linkage of the promoter to the coding sequence is a transcript fusion and expression of the encoded protein is desired, such a linkage is made such that the first translation initiation codon in the resulting transcript is the initiation codon of the coding sequence. Alternatively, if the linkage of the promoter to the coding sequence is a translational fusion and expression of the encoded protein is desired, the linkage is made such that the first translation initiation codon contained in the 5' untranslated sequence is linked to the promoter and is linked in such a way that the resulting translation product is in frame with the translational open reading frame encoding the desired protein. Nucleic acid sequences that may be "operably linked" include, but are not limited to: sequences that provide gene expression functions (i.e., gene expression elements such as promoters, 5 'untranslated regions, introns, protein coding regions, 3' untranslated regions, polyadenylation sites, and/or transcription terminators), sequences that provide DNA transfer and/or integration functions (i.e., T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), sequences that provide selective functions (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scorable marker functions, sequences that facilitate sequence manipulation in vitro or in vivo (i.e., polylinker sequences, site-specific recombination sequences), and sequences that provide replication functions (i.e., bacterial origins of replication, autonomously replicating sequences, centromeric sequences).

"pesticidal" or "pest-resistant" as used herein means toxic to crop pests, thereby achieving "control" and/or "control" of the crop pests. Preferably, the term "pesticidal" or "pest-resistant" refers to killing of a crop pest. More specifically, the target insect is an asian corn borer pest.

The plants, particularly corn, in the invention contain exogenous DNA in the genome, wherein the exogenous DNA comprises nucleotide sequences coding Cry1Ac protein and Cry2Ab protein, Asian corn borer pests are contacted with the Cry1Ac protein and the Cry2Ab protein by feeding plant tissues, the growth of the Asian corn borer is inhibited and/or death is caused after the contact, and meanwhile, the Asian corn borer can delay the generation of resistance to the Cry1Ac protein. Inhibition refers to lethal or sublethal. At the same time, the plant should be morphologically normal and can be cultured under conventional methods for consumption and/or production of the product. In addition, the plant may substantially eliminate the need for chemical or biological pesticides.

In the present invention, an exogenous DNA is introduced into a plant, such as a gene or expression cassette or recombinant vector encoding said Cry2Ab protein and/or said Cry1Ac protein, into a plant cell, and conventional transformation methods include, but are not limited to, agrobacterium-mediated transformation, microprojectile bombardment, direct uptake of DNA into protoplasts, electroporation, or whisker silicon-mediated DNA introduction.

The invention provides an insecticidal protein combination and a method for managing insect resistance, which have the following advantages:

1. resistance is delayed. The insects can generate resistance to a single Bt protein under continuous selection pressure, and the resistance of the Asian corn borers is effectively delayed or prevented by utilizing the two insecticidal proteins Cry1Ac and Cry2Ab, so that the control or prevention of the Asian corn borers is realized.

2. Effectively control resistant pests. The application of the Cry2Ab protein to the Asian corn borers which have developed resistance to the Cry1Ac protein can effectively control the resistant Asian corn borers into harmful plants, thereby ensuring that the plants are protected to a greater extent and the yield is stable.

3. The invention makes Cry1Ac protein and Cry2Ab protein express in plant body, and only needs to plant the transgenic plant without other measures, thereby saving a great deal of manpower, material resources and financial resources, and having stable and thorough effect.

The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.

Drawings

FIG. 1 is a schematic structural diagram of a recombinant expression vector Cry2Ab-pET30 containing a Cry2Ab nucleotide sequence for the insecticidal protein combinations and methods of the present invention for managing insect resistance;

FIG. 2 is a schematic of larval corrected mortality for ACB-Bts and ACB-AcR fed to Cry1Ac protein treated artificial feed for the insecticidal protein combinations of the present invention and methods of managing insect resistance;

FIG. 3 is a schematic of larval corrected mortality for ACB-BtS and ACB-AcR fed to Cry2Ab protein treated artificial feed for the insecticidal protein combinations of the present invention and methods of managing insect resistance;

FIG. 4 is a schematic representation of the larval growth inhibition ratio of ACB-BtS and ACB-AcR of artificial feed treated with a Cry1Ac protein fed with the insecticidal protein combinations of the present invention and methods of managing insect resistance;

FIG. 5 is a schematic representation of the larval growth inhibition rates of ACB-BtS and ACB-AcR in artificial feed treated with a Cry2Ab protein fed with the insecticidal protein combinations of the invention and methods of managing insect resistance.

Detailed Description

The technical scheme of the insecticidal protein combination and the method for managing insect resistance of the invention is further illustrated by the following specific examples.

First example, expression, purification and activation of Cry2Ab protein

1. Construction of recombinant expression vector containing Cry2Ab nucleotide sequence

The amino acid sequence (634 amino acids) of Cry2Ab insecticidal protein is shown as SEQ ID NO:1 in the sequence table; the nucleotide sequence of the Cry2Ab insecticidal protein is optimized according to the codon preference of Escherichia coli, and a Cry2Ab nucleotide sequence (1905 nucleotides) which encodes the amino acid sequence corresponding to the Cry2Ab insecticidal protein is obtained and is shown as SEQ ID NO:3 in the sequence table.

The Cry2Ab nucleotide sequence (shown as SEQ ID NO:3 in the sequence list) is synthesized by Nanjing Kingsley Biotech Co., Ltd, and the structural schematic diagram of the recombinant expression vector Cry2Ab-pET30 constructed by the nucleotide sequence is shown in FIG. 1 (wherein Kan represents a kanamycin resistance gene; f1 represents a replication origin of a phage f 1; Cry2Ab is a Cry2Ab nucleotide sequence (SEQ ID NO: 3); Lac I represents an operon; and ori represents a replication origin).

Then, the recombinant expression vector Cry2Ab-pET30 is transformed into competent cells of Escherichia coli T1 (Transgen, Beijing, China, CAT: CD501) by heat shock method under conditions of 50. mu.l of competent cells of Escherichia coli T1, 10. mu.l of plasmid DNA (recombinant expression vector Cry2Ab-pET30), water bath at 42 ℃ for 30 seconds, shaking culture at 37 ℃ for 1 hour (shaking at 100 rpm), ampicillin (100mg/L) coated with IPTG (isopropylthio- β -D-galactoside) and X-gal (5-bromo-4-chloro-3-indole- β -D-galactoside) on the surface (10 g/L of tryptone, 5 g/493 2 10/L of yeast extract, 15g/L of agar, pH adjusted to 7.5 with NaOH to grow white peptone), the white peptone is placed in LB liquid medium (10 g/L of tryptone, yeast extract 5 g/493 2 10/3, 5g/L of yeast extract, precipitated with NaOH at temperature of 10mM of 10.1205 min, precipitated with 10mM of EDTA, precipitated DNA, washed with 10mM of EDTA solution at room temperature of 10mM of 10M, precipitated DNA, diluted with 10M, dissolved in 10min, precipitated in 10.5.10M, dissolved in 10min, precipitated in 10M, precipitated in 10, diluted aqueous solution of EDTA (10) at room temperature of acetic acid, precipitated in 0.00, dried under conditions of 0.00, diluted with 10 mM) and mixed solution of 0.10 mM of 0.10, dissolved in 0.10 min, precipitated in 0.10 mM of acetic acid, dried overnight at room temperature, then precipitated glucose (10 mM) to obtain white peptone, then precipitated in 0.10.10 min, dried in 0.00, then, dried in 0.0.00, dried in 0.5 mM of glucose (10mM of acetic acid, dried under conditions of acetic acid, dried under 30. mu.0.10. mu.0.0.5 mM of glucose solution, dried supernatant, dried under 30min, 1min, dried overnight under 30min, 1min, and dried in 10mM of agar, dried in 0.10 min, and dried under 30min, and dried in.

After the extracted plasmid is subjected to enzyme digestion identification by KpnI and HindIII, sequencing verification is carried out on positive clones, and the result shows that the Cry2Ab nucleotide sequence inserted into the recombinant expression vector Cry2Ab-pET30 is the nucleotide sequence shown by SEQ ID NO. 3 in the sequence table, namely the Cry2Ab nucleotide sequence is correctly inserted.

2. Expression of Cry2Ab protein

The recombinant expression vector Cry2Ab-pET30 with correct sequencing is transformed into an escherichia coli expression host BL21(DE3) (purchased from Beijing Tiangen Biochemical technology Co., Ltd.), and the specific transformation method comprises the following steps: taking 50 μ L ice-thawed BL21(DE3) competent cells, adding plasmid DNA (recombinant expression vector Cry2Ab-pET30) and mixing gently, and standing on ice for 25 min; water bath at 42 deg.C for 90s, then quickly put back on ice and left standing for 2 min; adding 800 μ L LB liquid culture medium (tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, adjusting pH to 7.5 with NaOH) without antibiotic into the centrifuge tube, mixing, and recovering at 37 deg.C and 150rpm for 60 min; then, the cells were centrifuged at 4000rpm for 1min to collect the cells, and about 100. mu.L of the supernatant was gently blown and resuspended, and spread on LB plates (tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, agar 15g/L, pH adjusted to 7.5 with NaOH) containing 50. mu.g/mL kanamycin to grow overnight. After obtaining the expression strain, carrying out induction expression according to the following steps:

step 121, selecting a positive monoclonal containing recombinant expression vector Cry2Ab-pET30, shake culturing in 5mL LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, kanamycin 50. mu.g/mL, pH adjusted to 7.5 with NaOH) at 37 ℃ to make OD of the positive monoclonal₆₀₀The value reaches 0.5-0.6;

step 122, centrifuging 0.5mL of the bacterial liquid at the rotation speed of 12000rpm for 10min, and respectively taking supernatant and sediment as negative controls of non-induced expression;

step 123, adding isopropyl thiogalactoside (IPTG) into the residual 4.5mL of the bacterial liquid until the final concentration is 1mM, and continuing induction culture for 20h under the conditions of the rotating speed of 150rpm and the temperature of 16 ℃;

step 124, centrifuging the bacterial liquid after continuous induction for 10min at the rotation speed of 12000rpm, collecting thalli, and adding 10mM Tris (pH 8.0) for suspension;

step 125, ultrasonically crushing the suspended thalli, centrifuging at 12000rpm for 10min, respectively collecting supernatant and precipitate, and performing SDS-PAGE protein electrophoresis detection (refer to protein electrophoresis experiment (second edition)) on expression of Cry2Ab protein, wherein the detection result shows that: expression of Cry2Ab protein (72kDa) was observed in both supernatant and pellet.

3. Purification of Cry2Ab protein

The Cry2Ab protein is induced and expressed in large quantity according to the steps 121 and 125, and the collected crude protein is purified by a nickel column (His-Trap, FFGE Healthcare), which comprises the following steps:

step 131 of dissolving the bacterial cells collected in step 124 inBinding buffer (50mM NaH)₂PO₄500mM NaCl, 20mM imidazole, pH8.0), then placing on ice for ultrasonic crushing, centrifuging for 30min at the rotation speed of 12000rpm, collecting supernatant, and filtering the supernatant through a 0.45uM filter membrane to remove impurities;

step 132, balancing the nickel column with the binding buffer solution, washing 5 column volumes with a flow rate of 2 mL/min;

step 133, loading the filtered supernatant at a flow rate of 0.5 mL/min;

step 134, washing 5 column volumes with the binding buffer solution again at a flow rate of 2mL/min to enable Cry2Ab protein to be fully hung on the column;

step 135, continuously washing with the binding buffer solution to wash away foreign proteins at a flow rate of 2 mL/min;

step 136, elution buffer (50mM NaH)₂PO₄500mM NaCl, 500mM imidazole, pH8.0) were subjected to gradient elution (in order: eluting 3 column volumes by using the elution buffer solution with the concentration percentage of 10%, eluting 3 column volumes by using the elution buffer solution with the concentration percentage of 20%, eluting 3 column volumes by using the elution buffer solution with the concentration percentage of 40%, eluting 5 column volumes by using the elution buffer solution with the concentration percentage of 100%), wherein the flow rate is 2mL/min, respectively collecting an elution peak and an eluate, and carrying out SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) protein electrophoresis detection to obtain the purified Cry2Ab protein.

4. Activation of Cry2Ab protein

Dissolving the purified Cry2Ab protein in 50mM sodium carbonate buffer (pH 10.0); preparing trypsin into an aqueous solution with the concentration of 1mg/mL, and adding the aqueous solution of the trypsin into the Cry2Ab protein according to the mass ratio of 100:1(Cry2Ab protein: trypsin); after mixing, digesting for 1-3 hours at 37 ℃. And carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) protein electrophoresis detection on the activated Cry2Ab protein, wherein the result shows that the Cry2Ab protein after enzyme digestion activation is obtained.

The Cry1Ac protein used in the experiments described below was purchased from beijing leschen technologies ltd.

Second example, bioassay of Bt-sensitive Asiatic corn borer strains and Asiatic corn borer-resistant strains (Cry1Ac protein)

1. Source of test insects

The tested insects are Bt sensitive strains of Asiatic corn borer (Ostrinia furnacalis) (hereinafter ACB-Bts represents the Bt sensitive strains of the Asiatic corn borer) and Bt resistant strains of the Asiatic corn borer (Ostrinia furnacalis) (hereinafter ACB-AcR represents the Bt resistant strains of the Asiatic corn borer), and the insects of the two strains are from the institute of plant protection of the Chinese academy of agricultural sciences.

2. Asiatic corn borer bioassay method

Bioassays were performed using different concentrations for each Bt protein, determining concentrations of Cry1Ac protein ranging from 0 μ g/g to 50 μ g/g and Cry2Ab protein ranging from 0 μ g/g to 100 μ g/g. The preparation method of the Bt protein solution comprises the following steps: cry1Ac protein and distilled water (or buffer solution) are mixed according to a certain proportion to prepare Cry1Ac protein solutions with the concentrations of 0 mug/g, 0.01 mug/g, 0.05 mug/g, 0.1 mug/g, 0.5 mug/g, 1 mug/g, 5 mug/g, 10 mug/g and 50 mug/g respectively. Cry2Ab protein and distilled water (or buffer solution) are mixed according to a certain proportion to prepare Cry2Ab protein solutions with the concentrations of 0 mu g/g, 0.01 mu g/g, 0.05 mu g/g, 0.1 mu g/g, 0.5 mu g/g, 1 mu g/g, 5 mu g/g, 10 mu g/g, 50 mu g/g and 100 mu g/g respectively. Bt protein solutions (Cry1Ac protein solution or Cry2Ab protein solution) and artificial feed (the artificial feed is from plant protection research institute of Chinese academy of agricultural sciences) with different concentrations are mixed according to the weight ratio of 1.5: 1 separately mixing the mixed feeds, dispersing the mixed feeds in a bioassay plate of 48 wells, placing about 0.5g of the mixed feed in each well, and using as a blank an artificial feed treated with distilled water (or buffer) in an amount equivalent to the Bt protein solution (Cry1Ac protein solution or Cry2Ab protein solution). 1 young larva of asian corn borer (survival <24h) was inoculated on the mixed feed surface of each well of the bioassay plate, and the wells were covered with a sealing film. The bioassay plates were placed at 28 ℃ and 80% relative humidity with a photoperiod (light/dark) of 16:8 for 6 days, and larval mortality and larval growth inhibition were recorded starting at 7 days after inoculation. The combination of insect strains with Bt protein solution (Cry1Ac protein solution or Cry2Ab protein solution) concentrations was repeated 3 times per group, each repetition comprising 48 larvae.

Larval mortality is expressed as "actual mortality" (hereinafter simply mortality), and the calculated mortality takes into account actual dead larvae and surviving larvae that do not show significant weight gain (<0.1 mg/larva). The mortality rate of Asiatic corn borers is calculated by the following formula: mortality (%). 100 × [ number of dead larvae + number of surviving larvae showing no significant weight gain (<0.1 mg/larvae) ]/total number of insects tested. Mortality of each larva was corrected for mortality of larvae fed a blank control. Probability analysis was then performed on the corrected dose and mortality data, and the Bt protein concentration (Cry1Ac protein solution or Cry2Ab protein solution) that resulted in 50% (LC50) mortality and the corresponding 95% confidence interval were determined using POLO statistical software. The resistance ratio was calculated by dividing the LC50 value for ACB-AcR by the LC50 value for ACB-BtS.

The rate of inhibition of larval growth of the ostrinia furnacalis fed mixed diet was calculated using the formula larval growth inhibition (%) of 100 × (larval weight fed with blank control-larval weight fed with mixed diet)/(larval weight fed with blank control) — if there were no larvae with significant weight gain (<0.1 mg/larval), this repetition was designated as 100% larval growth inhibition.

3. Larva death rate and growth inhibition rate determination result of insect strain fed with mixed feed

The larva mortality of ACB-Bts and ACB-AcR of the mixed feed treated by feeding Cry1Ac protein is improved along with the increase of the concentration of the Cry1Ac protein, and the larva mortality of the ACB-Bts is obviously higher than that of the ACB-AcR at the same concentration. As shown in table 1 and fig. 2, the LC50 value calculated based on larval mortality of ACB-BtS was 0.21 μ g/g; the LC50 value calculated based on the larval mortality of ACB-AcR is greater than 1000 μ g/g, so the difference in LC50 values of ACB-BtS and ACB-AcR is very significant, as is the difference in resistance of the two to the Cry1Ac protein, i.e., the Cry1Ac protein has very weak insecticidal activity against ACB-AcR.

The larva mortality rate of ACB-Bts and ACB-AcR of the mixed feed treated by the Cry2Ab protein increases along with the increase of the concentration of the Cry2Ab protein, and the larva mortality rate of the ACB-Bts is higher than that of the ACB-AcR at the same concentration. As shown in table 1 and fig. 3, the LC50 value calculated based on larval mortality of ACB-BtS was 1.23 μ g/g; the LC50 value calculated based on the larval mortality of ACB-AcR was 6.71 μ g/g, so the difference in LC50 values for ACB-Bts and ACB-AcR was significant, but the Cry2Ab protein had insecticidal activity against both ACB-Bts and ACB-AcR.

As shown in figure 4, the abscissa is the logarithm of the concentration of Bt protein (mu g/g) used, the ordinate is the growth inhibition rate of the corresponding Asian corn borer, the larva growth inhibition rates of ACB-Bts and ACB-AcR fed with the Cry1Ac protein-treated mixed feed are both improved along with the increase of the concentration of the Cry1Ac protein, and the larva growth inhibition rate of the ACB-Bts at the same concentration is obviously higher than that of the ACB-AcR; when 70% growth inhibition rate is achieved, the concentration of Cry1Ac protein required by ACB-BtS is about 0.1 μ g/g, and the concentration of Cry1Ac protein required by ACB-AcR is about 500 μ g/g, indicating that Cry1Ac protein has weak inhibitory activity to ACB-AcR.

As shown in FIG. 5, the larva growth inhibition rate of both ACB-Bts and ACB-AcR fed with the Cry2Ab protein-treated mixed feed is increased along with the increase of the concentration of the Cry2Ab protein, and the larva growth inhibition rate of the ACB-Bts is higher than that of the ACB-AcR at the same concentration; when the growth inhibition rate of 100% is reached, the concentration of Cry2Ab protein required by ACB-Bts is about 1 mu g/g, and the concentration of Cry2Ab protein required by ACB-AcR is about 5 mu g/g, which shows that the Cry2Ab protein has equivalent inhibitory activity to ACB-Bts and ACB-AcR.

Table 1: toxicity results of Bt protein on ACB-Bts and ACB-AcR newborn larvae

The above results indicate that ACB-AcR has greater than 5000-fold resistance to Cry1Ac protein (i.e., resistance ratio greater than 5000), and Cry2Ab protein has insecticidal activity against both ACB-BtS and ACB-AcR, so ACB-AcR exhibits lower cross-resistance to Cry2Ab protein, with a significant reduction in resistance ratio to 5.4. Therefore, the Cry2Ab protein has low cross resistance with the Cry1Ac protein, the Cry2Ab protein and the Cry1Ac protein can delay or delay the resistance of the Asian corn borer to the Cry1Ac protein when being used in combination, and the Cry2Ab protein can effectively manage the resistance of the Asian corn borer species to the Cry1Ac protein.

Third example, use of Cry2Ab protein and Cry1Ac protein in the production of insect-resistant transgenic plants

As can be seen from the experimental results of the second example, the Cry2Ab protein and the Cry1Ac protein are expected to be useful for combined expression in transgenic plants such as corn plants to delay or prevent resistance to the plants by Asiatic corn borer.

The first method is a sequential transformation: wherein a plant transformed with a first gene (e.g., the Cry1Ac gene) is retransformed to introduce a second gene (e.g., the Cry2Ab gene). This sequential transformation preferably uses two different selectable marker genes, such as a kanamycin resistance gene and a phosphinothricin acetyltransferase gene (e.g., pat or bar gene) that confers resistance to glufosinate herbicide.

The second method is a co-transformation method: the nucleotide sequence encoding the Cry1Ac protein is expressed in plants along with the nucleotide sequence encoding the Cry2Ab protein, and plants comprising two selectable genes can be screened in their entirety by using selectable markers linked to the respective genes.

A third approach is an independent transformation event, with two insecticidal protein genes each being transferred separately into the genomes of different plants, which can then be combined in separate plants by crossing, and DNA tagging techniques can be used to select for plants containing these different genes.

In conclusion, the resistance of the Asiatic corn borers is effectively delayed or prevented by utilizing the two insecticidal proteins Cry1Ac and Cry2Ab, so that the control or prevention of the Asiatic corn borers is realized, the plants are protected to a greater extent, and the yield is stabilized; meanwhile, the Cry1Ac protein and the Cry2Ab protein are expressed in the plant body, and only the transgenic plant needs to be planted without adopting other measures, so that a great deal of manpower, material resources and financial resources are saved, and the effect is stable and thorough.

Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Beijing Dabei agricultural technology group, Inc

Beijing Dabei agricultural Biotechnology Co Ltd

<120> insecticidal protein combinations and methods for managing insect resistance

<130>DBNBC116

<160>4

<170>PatentIn version 3.5

<210>1

<211>634

<212>PRT

<213>Artificial Sequence

<220>

<223> Cry2Ab amino acid sequence

<400>1

Met Asp Asn Ser Val Leu Asn Ser Gly Arg Thr Thr Ile Cys Asp Ala

1 5 10 15

Tyr Asn Val Ala Ala His Asp Pro Phe Ser Phe Gln His Lys Ser Leu

20 25 30

Asp Thr Val Gln Lys Glu Trp Thr Glu Trp Lys Lys Asn Asn His Ser

35 40 45

Leu Tyr Leu Asp Pro Ile Val Gly Thr Val Ala Ser Phe Leu Leu Lys

50 55 60

Lys Val Gly Ser Leu Val Gly Lys Arg Ile Leu Ser Glu Leu Arg Asn

65 70 75 80

Leu Ile Phe Pro Ser Gly Ser Thr Asn Leu Met Gln Asp Ile Leu Arg

85 90 95

Glu Thr Glu Lys Phe Leu Asn Gln Arg Leu Asn Thr Asp Thr Leu Ala

100 105 110

Arg Val Asn Ala Glu Leu Thr Gly Leu Gln Ala Asn Val Glu Glu Phe

115 120 125

Asn Arg Gln Val Asp Asn Phe Leu Asn Pro Asn Arg Asn Ala Val Pro

130 135 140

Leu Ser Ile Thr Ser Ser Val Asn Thr Met Gln Gln Leu Phe Leu Asn

145 150 155 160

Arg Leu Pro Gln Phe Gln Met Gln Gly Tyr Gln Leu Leu Leu Leu Pro

165 170 175

Leu Phe Ala Gln Ala Ala Asn Leu His Leu Ser Phe Ile Arg Asp Val

180 185 190

Ile Leu Asn Ala Asp Glu Trp Gly Ile Ser Ala Ala Thr Leu Arg Thr

195 200 205

Tyr Arg Asp Tyr Leu Lys Asn Tyr Thr Arg Asp Tyr Ser Asn Tyr Cys

210 215 220

Ile Asn Thr Tyr Gln Ser Ala Phe Lys Gly Leu Asn Thr Arg Leu His

225 230 235 240

Asp Met Leu Glu Phe Arg Thr Tyr Met Phe Leu Asn Val Phe Glu Tyr

245 250 255

Val Ser Ile Trp Ser Leu Phe Lys Tyr Gln Ser Leu Leu Val Ser Ser

260 265 270

Gly Ala Asn Leu Tyr Ala Ser Gly Ser Gly Pro Gln Gln Thr Gln Ser

275 280 285

Phe Thr Ser Gln Asp Trp Pro Phe Leu Tyr Ser Leu Phe Gln Val Asn

290 295 300

Ser Asn Tyr Val Leu Asn Gly Phe Ser Gly Ala Arg Leu Ser Asn Thr

305 310 315 320

Phe Pro Asn Ile Val Gly Leu Pro Gly Ser Thr Thr Thr His Ala Leu

325 330 335

Leu Ala Ala Arg Val Asn Tyr Ser Gly Gly Ile Ser Ser Gly Asp Ile

340 345 350

Gly AlaSer Pro Phe Asn Gln Asn Phe Asn Cys Ser Thr Phe Leu Pro

355 360 365

Pro Leu Leu Thr Pro Phe Val Arg Ser Trp Leu Asp Ser Gly Ser Asp

370 375 380

Arg Glu Gly Val Ala Thr Val Thr Asn Trp Gln Thr Glu Ser Phe Glu

385 390 395 400

Thr Thr Leu Gly Leu Arg Ser Gly Ala Phe Thr Ala Arg Gly Asn Ser

405 410 415

Asn Tyr Phe Pro Asp Tyr Phe Ile Arg Asn Ile Ser Gly Val Pro Leu

420 425 430

Val Val Arg Asn Glu Asp Leu Arg Arg Pro Leu His Tyr Asn Glu Ile

435 440 445

Arg Asn Ile Ala Ser Pro Ser Gly Thr Pro Gly Gly Ala Arg Ala Tyr

450 455 460

Met Val Ser Val His Asn Arg Lys Asn Asn Ile His Ala Val His Glu

465 470 475 480

Asn Gly Ser Met Ile His Leu Ala Pro Asn Asp Tyr Thr Gly Phe Thr

485 490 495

Ile Ser Pro Ile His Ala Thr Gln Val Asn Asn Gln Thr Arg Thr Phe

500 505 510

Ile Ser Glu LysPhe Gly Asn Gln Gly Asp Ser Leu Arg Phe Glu Gln

515 520 525

Asn Asn Thr Thr Ala Arg Tyr Thr Leu Arg Gly Asn Gly Asn Ser Tyr

530 535 540

Asn Leu Tyr Leu Arg Val Ser Ser Ile Gly Asn Ser Thr Ile Arg Val

545 550 555 560

Thr Ile Asn Gly Arg Val Tyr Thr Ala Thr Asn Val Asn Thr Thr Thr

565 570 575

Asn Asn Asp Gly Val Asn Asp Asn Gly Ala Arg Phe Ser Asp Ile Asn

580 585 590

Ile Gly Asn Val Val Ala Ser Ser Asn Ser Asp Val Pro Leu Asp Ile

595 600 605

Asn Val Thr Leu Asn Ser Gly Thr Gln Phe Asp Leu Met Asn Ile Met

610 615 620

Leu Val Pro Thr Asn Ile Ser Pro Leu Tyr

625 630

<210>2

<211>1178

<212>PRT

<213>Artificial Sequence

<220>

<223> Cry1Ac amino acid sequence

<400>2

Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu

1 5 10 15

Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly

20 25 30

Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser

35 40 45

Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile

50 55 60

Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile

65 70 75 80

Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala

85 90 95

Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu

100 105 110

Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu

115 120 125

Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala

130 135 140

Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val

145 150 155 160

Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser

165 170 175

Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg

180 185 190

Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp His Ala Val

195 200 205

Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro Asp Ser Arg

210 215 220

Asp Trp Ile Arg Tyr Asn Gln Phe Arg Arg Glu Leu Thr Leu Thr Val

225 230 235 240

Leu Asp Ile Val Ser Leu Phe Pro Asn Tyr Asp Ser Arg Thr Tyr Pro

245 250 255

Ile Arg Thr Val Ser Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val

260 265 270

Leu Glu Asn Phe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu

275 280 285

Gly Ser Ile Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr

290 295 300

Ile Tyr Thr Asp Ala His Arg Gly Glu Tyr Tyr Trp Ser Gly His Gln

305 310 315 320

Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro

325 330 335

Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala

340 345 350

Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu Tyr Arg

355 360 365

Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser Val Leu Asp

370 375 380

Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu Pro Ser Ala Val

385 390 395 400

Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu Asp Glu Ile Pro Pro Gln

405 410 415

Asn Asn Asn Val Pro Pro Arg Gln Gly Phe Ser His Arg Leu Ser His

420 425 430

Val Ser Met Phe Arg Ser Gly Phe Ser Asn Ser Ser Val Ser Ile Ile

435 440 445

Arg Ala Pro Met Phe Ser Trp Ile His Arg Ser Ala Glu Phe Asn Asn

450 455 460

Ile Ile Ala Ser Asp Ser Ile Thr Gln Ile Pro Ala Val Lys Gly Asn

465 470 475 480

Phe Leu Phe Asn Gly Ser Val Ile Ser Gly Pro Gly Phe Thr Gly Gly

485 490 495

Asp Leu Val Arg Leu Asn Ser Ser Gly Asn Asn Ile Gln Asn Arg Gly

500 505 510

Tyr Ile Glu Val Pro Ile His Phe Pro Ser Thr Ser Thr Arg Tyr Arg

515 520 525

Val Arg Val Arg Tyr Ala Ser Val Thr Pro Ile His Leu Asn Val Asn

530 535 540

Trp Gly Asn Ser Ser Ile Phe Ser Asn Thr Val Pro Ala Thr Ala Thr

545 550 555 560

Ser Leu Asp Asn Leu Gln Ser Ser Asp Phe Gly Tyr Phe Glu Ser Ala

565 570 575

Asn Ala Phe Thr Ser Ser Leu Gly Asn Ile Val Gly Val Arg Asn Phe

580 585 590

Ser Gly Thr Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile Pro Val

595 600 605

Thr Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala Gln Lys Ala

610 615 620

Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly Leu Lys Thr Asn

625 630 635 640

Val Thr Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Thr Tyr Leu

645 650 655

Ser Asp Glu Phe Cys Leu Asp Glu Lys Arg Glu Leu Ser Glu Lys Val

660 665 670

Lys His Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Ser

675 680 685

Asn Phe Lys Asp Ile Asn Arg Gln Pro Glu Arg Gly Trp Gly Gly Ser

690 695 700

Thr Gly Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu Asn Tyr

705 710 715 720

Val Thr Leu Ser Gly Thr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr

725 730 735

Gln Lys Ile Asp Glu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu

740 745 750

Arg Gly Tyr Ile Glu Asp Ser Gln Asp Leu Glu Ile Tyr Ser Ile Arg

755 760 765

Tyr Asn Ala Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu

770 775 780

Trp Pro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn

785 790 795 800

Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys

805 810 815

Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp

820 825 830

Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val Trp Val

835 840 845

Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu

850 855 860

Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val

865 870 875 880

Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp

885 890 895

Glu Thr Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu

900 905 910

Phe Val Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala

915 920 925

Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr

930 935 940

Leu Pro Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu

945 950 955 960

Glu Leu Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg

965 970 975

Asn Val Ile Lys Asn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn

980 985 990

Val Lys Gly His Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val

995 1000 1005

Leu Val Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg

1010 1015 1020

Val Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys

1025 1030 1035

Glu Gly Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu Asn

1040 1045 1050

Asn Thr Asp Glu Leu Lys Phe Ser Asn Cys Val Glu Glu Glu Ile

1055 1060 1065

Tyr Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Val Asn Gln

1070 1075 1080

Glu Glu Tyr Gly Gly Ala Tyr Thr Ser Arg Asn Arg Gly Tyr Asn

1085 1090 1095

Glu Ala Pro Ser Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu

1100 1105 1110

Lys Ser Tyr Thr Asp Gly Arg Arg Glu Asn Pro Cys Glu Phe Asn

1115 1120 1125

Arg Gly Tyr Arg Asp Tyr Thr Pro Leu Pro Val Gly Tyr Val Thr

1130 1135 1140

Lys Glu Leu Glu Tyr Phe Pro Glu Thr Asp Lys Val Trp Ile Glu

1145 1150 1155

Ile Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Leu

1160 1165 1170

Leu Leu Met Glu Glu

1175

<210>3

<211>1905

<212>DNA

<213>Artificial Sequence

<220>

<223> Cry2Ab nucleotide sequence

<400>3

atggataact cagttctgaa ttcgggccgt accacgattt gcgatgccta taatgtcgcg 60

gcccatgacc cgtttagctt ccaacacaaa tctctggata ccgtgcagaa agaatggacg 120

gaatggaaga aaaacaatca tagcctgtac ctggacccga ttgtgggcac cgttgcgagc 180

tttctgctga agaaagtggg ctctctggtc ggtaaacgta ttctgagtga actgcgcaat 240

ctgatctttc cgtcaggttc gaccaacctg atgcaagata tcctgcgtga aacggaaaaa 300

ttcctgaacc agcgtctgaa taccgacacg ctggcgcgcg ttaacgccga actgaccggt 360

ctgcaagcaa acgtggaaga atttaatcgt caggttgata acttcctgaa cccgaatcgc 420

aacgctgtcc cgctgtctat taccagctct gtgaatacga tgcagcaact gtttctgaac 480

cgcctgccgc agttccaaat gcagggctat caactgctgc tgctgccgct gtttgcgcag 540

gcagctaatc tgcatctgag cttcattcgt gatgtgatcc tgaacgccga cgaatggggt 600

atttctgcgg ccaccctgcg tacgtatcgc gattacctga aaaattatac ccgcgactat 660

tccaactact gcatcaatac gtaccagtca gcatttaaag gcctgaatac ccgtctgcac 720

gatatgctgg aattccgcac ctatatgttt ctgaacgtgt tcgaatacgt tagcatttgg 780

tctctgttta aatatcagag cctgctggtt agctccggtg caaacctgta cgctagcggc 840

tctggtccgc agcaaaccca aagtttcacg tcccaggatt ggccgtttct gtattcactg 900

ttccaggtca attcgaacta cgtgctgaat ggctttagtg gtgcccgtct gtccaacacc 960

ttcccgaata tcgtgggtct gccgggttca accacgaccc acgcactgct ggcagctcgt 1020

gttaactatt cgggcggtat ttcatcgggc gatatcggtg cctcgccgtt taatcagaac 1080

ttcaattgta gcacctttct gccgccgctg ctgacgccgt tcgttcgtag ctggctggat 1140

agtggttccg accgtgaagg tgtcgcaacc gtgacgaatt ggcagaccga atcttttgaa 1200

acgaccctgg gcctgcgtag tggtgcattc accgctcgcg gcaactccaa ctacttcccg 1260

gattacttca tccgtaacat cagcggcgtg ccgctggttg tgcgcaacga agacctgcgt 1320

cgcccgctgc attataacga aattcgcaat atcgcctcac cgtcgggcac cccgggcggt 1380

gcacgtgcat acatggtttc tgtccacaac cgcaaaaaca atattcatgc ggtccacgaa 1440

aatggcagta tgatccatct ggccccgaac gattataccg gttttacgat ttccccgatc 1500

cacgcaaccc aagtgaacaa tcagacccgt acgtttattt cagaaaaatt cggcaatcag 1560

ggtgattcgc tgcgctttga acagaacaat acgaccgctc gttataccct gcgcggcaac 1620

ggtaatagtt ataacctgta cctgcgtgtt agctctatcg gcaattccac cattcgtgtt 1680

acgatcaacg gtcgcgtcta caccgcgacg aacgtgaata cgaccacgaa caatgatggc 1740

gtgaacgaca atggtgcacg ctttagcgat attaacatcg gcaatgtcgt ggctagttcc 1800

aatagcgatg ttccgctgga cattaacgtc accctgaatt ctggtacgca gtttgacctg 1860

atgaatatta tgctggtgcc gaccaacatc agcccgctgt attaa 1905

<210>4

<211>3537

<212>DNA

<213>Artificial Sequence

<220>

<223> Cry1Ac nucleotide sequence

<400>4

atggacaaca acccaaacat caacgaatgc attccataca actgcttgag taacccagaa 60

gttgaagtac ttggtggaga acgcattgaa accggttaca ctcccatcga catctccttg 120

tccttgacac agtttctgct cagcgagttc gtgccaggtg ctgggttcgt tctcggacta 180

gttgacatca tctggggtat ctttggtcca tctcaatggg atgcattcct ggtgcaaatt 240

gagcagttga tcaaccagag gatcgaagag ttcgccagga accaggccat ctctaggttg 300

gaaggattga gcaatctcta ccaaatctat gcagagagct tcagagagtg ggaagccgat 360

cctactaacc cagctctccg cgaggaaatg cgtattcaat tcaacgacat gaacagcgcc 420

ttgaccacag ctatcccatt gttcgcagtc cagaactacc aagttcctct cttgtccgtg 480

tacgttcaag cagctaatct tcacctcagc gtgcttcgag acgttagcgt gtttgggcaa 540

aggtggggat tcgatgctgc aaccatcaat agccgttaca acgaccttac taggctgatt 600

ggaaactaca ccgaccacgc tgttcgttgg tacaacactg gcttggagcg tgtctggggt 660

cctgattcta gagattggat tagatacaac cagttcagga gagaattgac cctcacagtt 720

ttggacattg tgtctctctt cccgaactat gactccagaa cctaccctat ccgtacagtg 780

tcccaactta ccagagaaat ctatactaac ccagttcttg agaacttcga cggtagcttc 840

cgtggttctg cccaaggtat cgaaggctcc atcaggagcc cacacttgat ggacatcttg 900

aacagcataa ctatctacac cgatgctcac agaggagagt attactggtc tggacaccag 960

atcatggcct ctccagttgg attcagcggg cccgagttta cctttcctct ctatggaact 1020

atgggaaacg ccgctccaca acaacgtatc gttgctcaac taggtcaggg tgtctacaga 1080

accttgtctt ccaccttgta cagaagaccc ttcaatatcg gtatcaacaa ccagcaactt 1140

tccgttcttg acggaacaga gttcgcctat ggaacctctt ctaacttgcc atccgctgtt 1200

tacagaaaga gcggaaccgt tgattccttg gacgaaatcc caccacagaa caacaatgtg 1260

ccacccaggc aaggattctc ccacaggttg agccacgtgt ccatgttccg ttccggattc 1320

agcaacagtt ccgtgagcat catcagagct cctatgttct cttggataca tcgtagtgct 1380

gagttcaaca acatcatcgc atccgatagt attactcaaa tccctgcagt gaagggaaac 1440

tttctcttca acggttctgt catttcagga ccaggattca ctggtggaga cctcgttaga 1500

ctcaacagca gtggaaataa cattcagaat agagggtata ttgaagttcc aattcacttc 1560

ccatccacat ctaccagata tagagttcgt gtgaggtatg cttctgtgac ccctattcac 1620

ctcaacgttaattggggtaa ttcatccatc ttctccaata cagttccagc tacagctacc 1680

tccttggata atctccaatc cagcgatttc ggttactttg aaagtgccaa tgcttttaca 1740

tcttcactcg gtaacatcgt gggtgttaga aactttagtg ggactgcagg agtgattatc 1800

gacagattcg agttcattcc agttactgca acactcgagg ctgagtacaa ccttgagaga 1860

gcccagaagg ctgtgaacgc cctctttacc tccaccaatc agcttggctt gaaaactaac 1920

gttactgact atcacattga ccaagtgtcc aacttggtca cctaccttag cgatgagttc 1980

tgcctcgacg agaagcgtga actctccgag aaagttaaac acgccaagcg tctcagcgac 2040

gagaggaatc tcttgcaaga ctccaacttc aaagacatca acaggcagcc agaacgtggt 2100

tggggtggaa gcaccgggat caccatccaa ggaggcgacg atgtgttcaa ggagaactac 2160

gtcaccctct ccggaacttt cgacgagtgc taccctacct acttgtacca gaagatcgat 2220

gagtccaaac tcaaagcctt caccaggtat caacttagag gctacatcga agacagccaa 2280

gaccttgaaa tctactcgat caggtacaat gccaagcacg agaccgtgaa tgtcccaggt 2340

actggttccc tctggccact ttctgcccaa tctcccattg ggaagtgtgg agagcctaac 2400

agatgcgctc cacaccttga gtggaatcct gacttggact gctcctgcag ggatggcgag 2460

aagtgtgccc accattctca tcacttctcc ttggacatcg atgtgggatg tactgacctg 2520

aatgaggacc tcggagtctg ggtcatcttc aagatcaaga cccaagacgg acacgcaaga 2580

cttggcaacc ttgagtttct cgaagagaaa ccattggtcg gtgaagctct cgctcgtgtg 2640

aagagagcag agaagaagtg gagggacaaa cgtgagaaac tcgaatggga aactaacatc 2700

gtttacaagg aggccaaaga gtccgtggat gctttgttcg tgaactccca atatgatcag 2760

ttgcaagccg acaccaacat cgccatgatc cacgccgcag acaaacgtgt gcacagcatt 2820

cgtgaggctt acttgcctga gttgtccgtg atccctggtg tgaacgctgc catcttcgag 2880

gaacttgagg gacgtatctt taccgcattc tccttgtacg atgccagaaa cgtcatcaag 2940

aacggtgact tcaacaatgg cctcagctgc tggaatgtga aaggtcatgt ggacgtggag 3000

gaacagaaca atcagcgttc cgtcctggtt gtgcctgagt gggaagctga agtgtcccaa 3060

gaggttagag tctgtccagg tagaggctac attctccgtg tgaccgctta caaggaggga 3120

tacggtgagg gttgcgtgac catccacgag atcgagaaca acaccgacga gcttaagttc 3180

tccaactgcg tcgaggaaga aatctatccc aacaacaccg ttacttgcaa cgactacact 3240

gtgaatcagg aagagtacgg aggtgcctac actagccgta acagaggtta caacgaagct 3300

ccttccgttc ctgctgacta tgcctccgtg tacgaggaga aatcctacac agatggcaga 3360

cgtgagaacc cttgcgagtt caacagaggt tacagggact acacaccact tccagttggc 3420

tatgttacca aggagcttga gtactttcct gagaccgaca aagtgtggat cgagatcggt 3480

gaaaccgagg gaaccttcat cgtggacagc gtggagcttc tcttgatgga ggaataa 3537

Claims

1. A method for managing insect resistance is characterized by comprising the step of contacting Asian corn borer with at least Cry2Ab protein and Cry1Ac protein, wherein the amino acid sequence of the Cry2Ab protein is shown as SEQ ID NO. 1, and the amino acid sequence of the Cry1Ac protein is shown as SEQ ID NO. 2.

2. The method of managing insect resistance according to claim 1, wherein said Cry2Ab protein and Cry1Ac protein are present in a bacterium or transgenic plant that produces at least said Cry2Ab protein and Cry1Ac protein, said asian corn borer is contacted with at least said Cry2Ab protein and said Cry1Ac protein by ingestion of a tissue of said bacterium or said transgenic plant, upon which said asian corn borer growth is inhibited and/or caused to die, to achieve management of resistance to asian corn borer.

3. The method of managing insect resistance according to claim 1, wherein asian corn borer resistant to a Cry1Ac protein is contacted with at least said Cry2Ab protein, said Cry2Ab protein is present in a bacterium or transgenic plant that produces at least said Cry2Ab protein, said asian corn borer resistant to a Cry1Ac protein is contacted with at least said Cry2Ab protein by ingestion of a tissue of said bacterium or said transgenic plant, and growth of said asian corn borer resistant to a Cry1Ac protein is inhibited and/or caused to die upon contact to achieve management of resistance of said asian corn borer resistant to a Cry1Ac protein.

4. The method of managing insect resistance according to claim 1, wherein asian corn borer resistant to a Cry2Ab protein is contacted with at least said Cry1Ac protein, said Cry1Ac protein is present in a bacterium or transgenic plant that produces at least said Cry1Ac protein, said asian corn borer resistant to a Cry2Ab protein is contacted with at least said Cry1Ac protein by ingestion of a tissue of said bacterium or said transgenic plant, and growth of said asian corn borer resistant to a Cry2Ab protein is inhibited and/or caused to die upon contact to achieve management of resistance of said asian corn borer resistant to a Cry2Ab protein.

5. The method of managing insect resistance according to any one of claims 2 to 4, wherein the transgenic plant is at any stage of growth.

6. The method of managing insect resistance according to any one of claims 2-4, wherein the tissue of the transgenic plant is a root, leaf, stem, fruit, tassel, ear, anther, or filament.

7. The method of managing insect resistance according to any one of claims 2 to 4, wherein the control of Asian corn borer endangered plants is not altered by changes in planting location and/or planting time.

8. The method of managing insect resistance according to claim 7, wherein the plant is maize, wheat, sorghum, millet, rice or soybean.

9. The method for managing insect resistance according to claim 8, wherein the nucleotide sequence of the Cry2Ab protein is set forth in SEQ ID No. 3.

10. The method for managing insect resistance according to claim 8 or 9, characterized in that the nucleotide sequence of the Cry1Ac protein is set forth in SEQ ID No. 4.

11. The method of managing insect resistance according to claim 6, wherein the plant is maize, wheat, sorghum, millet, rice or soybean.

12. The method for managing insect resistance according to claim 11, wherein the nucleotide sequence of the Cry2Ab protein is set forth in SEQ ID No. 3.

13. The method for managing insect resistance according to claim 11 or 12, characterized in that the nucleotide sequence of the Cry1Ac protein is set forth in SEQ ID No. 4.

14. The method of managing insect resistance according to claim 5, wherein the plant is maize, wheat, sorghum, millet, rice or soybean.

15. The method for managing insect resistance according to claim 14, wherein the nucleotide sequence of the Cry2Ab protein is set forth in SEQ ID No. 3.

16. The method for managing insect resistance according to claim 14 or 15, characterized in that the nucleotide sequence of the Cry1Ac protein is set forth in SEQ ID No. 4.

17. The method of managing insect resistance according to any one of claims 2-4, wherein the plant is maize, wheat, sorghum, millet, rice or soybean.

18. The method for managing insect resistance according to claim 17, wherein the nucleotide sequence of the Cry2Ab protein is set forth in SEQ ID No. 3.

19. The method for managing insect resistance according to claim 17, wherein the nucleotide sequence of the Cry1Ac protein is set forth in SEQ ID No. 4.

20. The method for managing insect resistance according to claim 18, wherein the nucleotide sequence of the Cry1Ac protein is set forth in SEQ ID No. 4.

21. The method for managing insect resistance according to claim 7, wherein the nucleotide sequence of the Cry2Ab protein is set forth in SEQ ID NO. 3.

22. The method for managing insect resistance according to claim 21, wherein the nucleotide sequence of the Cry1Ac protein is set forth in SEQ ID No. 4.

23. The method for managing insect resistance according to claim 6, wherein the nucleotide sequence of the Cry2Ab protein is set forth in SEQ ID NO. 3.

24. The method for managing insect resistance according to claim 23, wherein the nucleotide sequence of the Cry1Ac protein is set forth in SEQ ID No. 4.

25. The method for managing insect resistance according to claim 5, wherein the nucleotide sequence of the Cry2Ab protein is set forth in SEQ ID NO. 3.

26. The method for managing insect resistance according to claim 25, wherein the nucleotide sequence of the Cry1Ac protein is set forth in SEQ ID No. 4.

27. The method for managing insect resistance according to any one of claims 1 to 4, wherein the nucleotide sequence of the Cry2Ab protein is represented by SEQ ID NO. 3.

28. The method for managing insect resistance according to claim 27, wherein the nucleotide sequence of the Cry1Ac protein is set forth in SEQ ID No. 4.

29. The method for managing insect resistance according to claim 7, wherein the nucleotide sequence of the Cry1Ac protein is set forth in SEQ ID NO. 4.

30. The method for managing insect resistance according to claim 6, wherein the nucleotide sequence of the Cry1Ac protein is set forth in SEQ ID NO. 4.

31. The method for managing insect resistance according to claim 5, wherein the nucleotide sequence of the Cry1Ac protein is set forth in SEQ ID NO. 4.

32. The method for managing insect resistance according to any one of claims 1 to 4, wherein the nucleotide sequence of the Cry1Ac protein is shown as SEQ ID NO. 4.

33. The method for controlling the Asiatic corn borers is characterized by comprising the step of contacting the Asiatic corn borers with at least Cry2Ab protein and Cry1Ac protein, so that the control of the Asiatic corn borers is realized, wherein the amino acid sequence of the Cry2Ab protein is shown as SEQ ID NO:1, and the amino acid sequence of the Cry1Ac protein is shown as SEQ ID NO: 2.

34. The method for controlling Asian corn borer as claimed in claim 33, wherein Cry2Ab protein and Cry1Ac protein are present in a bacterium or transgenic plant producing at least said Cry2Ab protein and Cry1Ac protein, said Asian corn borer contacting at least said Cry2Ab protein and Cry1Ac protein by ingesting tissue from said bacterium or said transgenic plant, said Asian corn borer growth being inhibited and/or caused to die upon contact, to effect control of Asian corn borer harm plants.

35. The method for controlling asian corn borer according to claim 33, wherein asian corn borer resistant to Cry1Ac protein is contacted with at least said Cry2Ab protein, said Cry2Ab protein is present in a bacterium or transgenic plant producing at least said Cry2Ab protein, said asian corn borer resistant to Cry1Ac protein is contacted with at least said Cry2Ab protein by ingestion of a tissue of said bacterium or said transgenic plant, and growth of said asian corn borer resistant to Cry1Ac protein is inhibited and/or caused to die after contact, to effect control of said asian corn borer resistant to Cry1Ac protein.

36. The method for controlling asian corn borer according to claim 33, wherein asian corn borer resistant to Cry2Ab protein is contacted with at least said Cry1Ac protein, said Cry1Ac protein is present in a bacterium or transgenic plant producing at least said Cry1Ac protein, said asian corn borer resistant to Cry2Ab protein is contacted with at least said Cry1Ac protein by ingestion of a tissue of said bacterium or said transgenic plant, and growth of said asian corn borer resistant to Cry2Ab protein is inhibited and/or caused to die after contact, to effect control of said asian corn borer resistant to Cry2Ab protein.

37. The method of controlling asian corn borer according to any of claims 34 to 36, wherein said transgenic plant is at any stage of growth.

38. The method of controlling asian corn borer according to any one of claims 34 to 36, wherein said tissue of transgenic plant is root, leaf, stem, fruit, tassel, ear, anther or filament.

39. The method of controlling asian corn borer according to any one of claims 34 to 36, wherein said control of asian corn borer dangerous plants is not altered by changes in planting location and/or planting time.

40. The method of controlling asian corn borer as claimed in claim 39 wherein said plant is corn, wheat, sorghum, millet, rice or soybean.

41. The method for controlling Asian corn borer according to claim 40, wherein the Cry2Ab protein has the nucleotide sequence as shown in SEQ ID NO. 3.

42. The method for controlling Asian corn borer according to claim 40 or 41, wherein the Cry1Ac protein has the nucleotide sequence shown in SEQ ID NO. 4.

43. The method of controlling asian corn borer as claimed in claim 38, wherein said plant is corn, wheat, sorghum, millet, rice or soybean.

44. The method for controlling Asian corn borer according to claim 43, wherein the Cry2Ab protein has the nucleotide sequence as shown in SEQ ID NO. 3.

45. The method for controlling Asian corn borer according to claim 43 or 44, wherein the Cry1Ac protein has the nucleotide sequence as shown in SEQ ID NO. 4.

46. The method of controlling asian corn borer as claimed in claim 37 wherein said plant is corn, wheat, sorghum, millet, rice or soybean.

47. The method for controlling Asian corn borer as claimed in claim 46, wherein the nucleotide sequence of Cry2Ab protein is shown in SEQ ID NO. 3.

48. The method for controlling Asian corn borer according to claim 46 or 47, wherein the Cry1Ac protein has the nucleotide sequence as shown in SEQ ID NO. 4.

49. The method of controlling asian corn borer according to any one of claims 34 to 36, wherein said plant is corn, wheat, sorghum, millet, rice or soybean.

50. The method for controlling Asian corn borer as claimed in claim 49, wherein the Cry2Ab protein has the nucleotide sequence as shown in SEQ ID NO. 3.

51. The method for controlling Asian corn borer as claimed in claim 49, wherein the nucleotide sequence of Cry1Ac protein is shown in SEQ ID NO. 4.

52. The method for controlling Asian corn borer according to claim 50, wherein the Cry1Ac protein has the nucleotide sequence as shown in SEQ ID NO. 4.

53. The method for controlling Asian corn borer as claimed in claim 39, wherein the Cry2Ab protein has the nucleotide sequence as shown in SEQ ID NO. 3.

54. The method for controlling Asian corn borer according to claim 53, wherein the Cry1Ac protein has the nucleotide sequence as shown in SEQ ID NO. 4.

55. The method for controlling Asian corn borer according to claim 38, wherein the Cry2Ab protein has the nucleotide sequence as shown in SEQ ID NO. 3.

56. The method for controlling Asian corn borer as claimed in claim 55, wherein the Cry1Ac protein has the nucleotide sequence as shown in SEQ ID NO. 4.

57. The method for controlling Asian corn borer according to claim 37, wherein the Cry2Ab protein has the nucleotide sequence as shown in SEQ ID NO. 3.

58. The method for controlling Asian corn borer as claimed in claim 57, wherein the Cry1Ac protein has the nucleotide sequence as shown in SEQ ID NO. 4.

59. The method for controlling Asian corn borer according to any one of claims 33-36, wherein the Cry2Ab protein has the nucleotide sequence as shown in SEQ ID NO. 3.

60. The method for controlling Asian corn borer as claimed in claim 59, wherein the Cry1Ac protein has the nucleotide sequence as shown in SEQ ID NO. 4.

61. The method for controlling Asian corn borer as claimed in claim 39, wherein the Cry1Ac protein has the nucleotide sequence as shown in SEQ ID NO. 4.

62. The method for controlling Asian corn borer according to claim 38, wherein the Cry1Ac protein has the nucleotide sequence as shown in SEQ ID NO. 4.

63. The method for controlling Asian corn borer according to claim 37, wherein the Cry1Ac protein has the nucleotide sequence as shown in SEQ ID NO. 4.

64. The method for controlling Asian corn borer according to any one of claims 33-36, wherein the Cry1Ac protein has the nucleotide sequence as shown in SEQ ID NO. 4.

65. The application of the Cry2Ab protein and the Cry1Ac protein in combination to preventing or delaying the generation of resistance to the Cry1Ac protein or the Cry2Ab protein in Asian corn borer population, wherein the amino acid sequence of the Cry2Ab protein is shown as SEQ ID NO:1, and the amino acid sequence of the Cry1Ac protein is shown as SEQ ID NO: 2.

66. The application of the Cry2Ab protein and the Cry1Ac protein in combination to control Asian corn borer population generating resistance to the Cry1Ac protein or the Cry2Ab protein, wherein the amino acid sequence of the Cry2Ab protein is shown as SEQ ID NO:1, and the amino acid sequence of the Cry1Ac protein is shown as SEQ ID NO: 2.