CN111606984A

CN111606984A - Plant insect-resistant protein and coding gene and application thereof

Info

Publication number: CN111606984A
Application number: CN202010426563.2A
Authority: CN
Inventors: 贾志伟; 李晓娇; 吕玉平; 刘枫; 王强; 李树秀; 孙宇; 李涛; 赵丽媛; 张原�
Original assignee: Longping Biotechnology Hainan Co ltd
Current assignee: Longping Biotechnology Hainan Co ltd
Priority date: 2020-05-19
Filing date: 2020-05-19
Publication date: 2020-09-01
Anticipated expiration: 2040-05-19
Also published as: CN111606984B

Abstract

The application discloses a protein for resisting insects of plants, and the amino acid sequence of the protein is SEQ ID NO. 3. Also provides the coding gene SEQ ID NO. 1 of the insect-resistant protein. The invention further provides application of the insect-resistant protein and the insect-resistant gene in anti-cutworm. The insect-resistant protein and the insect-resistant gene provided by the invention can provide better cutworm resistance and spodoptera frugiperda resistance.

Description

Plant insect-resistant protein and coding gene and application thereof

Technical Field

The application relates to the technical field of genetic engineering biological control, in particular to an artificially modified insect-resistant gene mCry1Fa, a protein formed by coding and expressing the same and application of the gene in the aspect of insect resistance of plants.

Background

Biological control is to control the population quantity of pests by using some beneficial organisms or biological metabolites to achieve the purpose of reducing or eliminating the pests, such as trichogramma or beauveria bassiana to control the meadow moth. It is characterized by safety to human and livestock, little pollution to environment and long-term control of certain pests; but the effect is often unstable and the same investment is required to be made no matter the weight of the meadow moth is light. In order to solve the limitation of agricultural control, chemical control, physical control and biological control in practical application, scientists find that some insect-resistant transgenic plants can be obtained to prevent and control plant pests by transferring insect-resistant genes for coding insecticidal proteins into plants through research.

The Bt gene codes insecticidal crystal protein and is from Bacillus thuringiensis (Bacillus thuringiensis), which is gram-positive agrobacterium and belongs to Bacillus, and the Bacillus is short rod-shaped, flagellar, single or short chain-forming. It produces insecticidal companion cell crystal proteins called endotoxins (genes controlling the synthesis of such proteins on plasmids) during sporulation, which have specific insecticidal activity against a wide variety of insects of the Lepidoptera, Diptera, Coleoptera, Hymenoptera, and the like.

In 1981, Schenpf and Whiteley cloned the first Cry gene Cry1Aa1 encoding endotoxin from Bacillus thuringiensis (Bacillus thuringiensis). In 1985, Adang, M.J and the like cloned Cry1Ac gene from Bacillus thuringiensis (Bacillus thuringiensis), in 1986, Wabiko, H. and the like cloned Cry1Ab gene from Bacillus thuringiensis, and the gene codes 1155 amino acids and is an insect-resistant gene which is widely applied in industrialization at present. In 1991 Chambers, J cloned the cry1Fa gene. By far, more than one thousand species of Bacillus thuringiensis strains and more than ten thousand species of insecticidal crystal proteins have been identified.

There are multiple instars in the development of cutworms as larvae. While biting seedlings by later instar larvae produces the most significant damage and economic losses, leaf feeding often results in reduced yield in crops such as corn. Near late instars (e.g., third to fourth instars), the larvae begin to bite into the plant or plant part, particularly the seedling. Populations that cause economic losses can be unpredictably formed with few early warning signs due to changes in feeding patterns. Big cutworms can destroy several seedlings per day, and severe pest infestation can remove entire stands of crop. The night habit of cutworms and the act of digging holes into the ground also make observation and management problematic.

Cutworms (The black cutwork) (Agrotis ipsilon (Hufnagel); Lepidoptera (Lepidoptera): Noctuidae) are serious pests of a variety of crops including corn, cotton, Brassica vegetables (chinese cabbage (Brassica), broccoli, cabbage, chinese cabbage) and turf. Secondary host plants include beetroot, capsicum, chickpea, faba beans, lettuce, alfalfa (lucerne), onion, potato, radish, rape, rice, soybean, strawberry, sugar beet, tobacco, tomato, and forest trees. In north america, pests of the genus antiarthia (Agrotis) feed on alfalfa, corn (maize), tobacco, hemp, onions, strawberries, blackberries, raspberries, alfalfa (alfalfalfa), barley, beans, cabbage, oats, peas, potatoes, sweet potatoes (sweet potatoes), tomatoes, garden flowers, grasses, alfalfa (lucern), corn (maize), asparagus, grapes, almost all types of leaves, wild grasses and many other crops and garden plants.

Culture controls such as peripheral weed control on cutworms (a. ipsilon) may help prevent serious attacks; however, this approach is not always applicable or effective. Infestation is sporadic and the application of pesticides prior to or at the time of planting has been ineffective in the past. There are some baits to control cutworms in crops. To protect turfgrass such as careengbentgrass, chemical insecticides are used. The use of chemical pesticides in turf-covered areas (such as golf greens, sports fields, parks and other recreational areas, professional landscaping, and home lawns) is of particular interest because of the close public contact with these areas. Natural products such as nematodes and azadirachtin (azadirachtin) are often not effective. The rapid feeding and hole digging actions of cutworms make them particularly difficult to control using current bio-based pest solutions. For example, Cry1A (b) toxins are essentially ineffective against cutworms. Chinese patent application publication No. CN102246823A discloses that Cry1F gene is modified to have its encoded protein kill effect.

Spodoptera frugiperda (academic name: Spodoptera frugiperda): is a kind of moth of spodoptera of the family Spodoptera. Adults live at night, laying approximately 100 eggs on top of the plant leaves, and the egg stage is at a temperature of 25 ℃ for 3 days. Newly hatched larvae feed on the egg shell itself and then stand for 2-10 hours. The larvae, caterpillars, prefer to feed on new leaves, and due to their feeding habits, usually find one new leaf each, with their larvae feeding on corn and rice, respectively. In the prior art, chemical control methods are mainly adopted for controlling spodoptera frugiperda at present, and the spodoptera frugiperda has strong pesticide resistance to a plurality of pesticides. In addition, the problems of pesticide residue and the like caused by a chemical prevention and control mode exist in the risks of potential safety hazards of people and animals and ecological environment influence. The method is imperative to search for green, safe, efficient and continuous measures for preventing and controlling Spodoptera frugiperda.

Disclosure of Invention

The invention aims to provide a modified Cry1Fa gene, and after the mCry1Fa gene provided by the invention is transferred into a plant, the obtained transgenic plant with high expression mCry1Fa protein has higher cutworm and/or Spodoptera frugiperda resistance.

The invention discloses a protein for resisting insects of plants, and the amino acid sequence of the protein is SEQ ID NO. 3.

The invention also discloses an insect-resistant gene for coding the plant insect-resistant protein, and particularly the nucleotide sequence of the insect-resistant gene is SEQ ID NO. 1. After the mCry1Fa gene is transferred into a plant, the obtained transgenic plant with high expression mCry1Fa protein has higher resistance to cutworm and/or spodoptera frugiperda.

The invention further provides an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the insect-resistant gene.

In another aspect of the present invention, there is provided an expression vector, wherein the expression vector comprises the insect-resistant gene described above.

In one embodiment of the present invention, the expression vector comprises the following gene structures in sequence:

from the maize ubiquitin gene promoter (Ubi); the insect-resistant gene of

claim

2 or 3; a nopaline synthase terminator (Nos); encoding a phosphinothricin acetyltransferase gene (PAT); terminator from cauliflower mosaic virus (CaMV) (35 s).

The invention also provides a protein for resisting the insect of the plant, or the insect-resistant gene, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the insect-resistant gene, wherein the application is selected from one or two of the following: a) Preparing a medicament having an effect of resisting cutworm and/or spodoptera frugiperda; b) cultivating transgenic plants having or having increased resistance to cutworm and spodoptera frugiperda.

The invention further provides a method for cultivating plants with or improved capacity of resisting cutworm and/or spodoptera frugiperda, which is characterized by comprising the following steps: and (3) introducing the insect-resistant gene into a receptor plant to obtain a transgenic plant.

In one embodiment according to the invention, the plant is selected from one or more of monocotyledons, dicotyledons, gramineae; preferably corn.

The invention has the following beneficial effects:

the killing efficiency of the mCry1Fa on cutworms and/or spodoptera frugiperda provided by the invention is far higher than that of Cry1Fa, the modified gene mCry1Fa has better insecticidal effect, and plants expressing the mCry1Fa gene also have better resistance to cutworms and/or spodoptera frugiperda, thereby being beneficial to the control of cutworms and/or spodoptera frugiperda in agriculture.

Drawings

FIG. 1 is a flow chart of the construction of the recombinant cloning vector LP05-T containing mCry1Fa nucleotide sequence according to the present invention;

FIG. 2 is a flow chart of construction of recombinant expression vector LP-PT05 containing mCry1Fa nucleotide sequence according to the present invention;

FIG. 3 is a PCR assay of transformants of the invention, wherein WT is a wild-type plant, PC is a plasmid control, NC is a water control, and 1-20 are 20 positive transformants;

fig. 4 is a corn borer resistance bioassay map of transformants of the method of controlling pests of the present invention, wherein WT is a wild type plant, Cry1Fa is transformed with an unmodified Cry1Fa transformation event, mCry1Fa is transformed with a modified Cry1Fa transformation event.

Detailed Description

The following examples are intended to illustrate the present application but are not intended to limit the scope of the present application.

Specific embodiments of the present application will be described in more detail below. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

Example 1 Cry1Fa Gene acquisition and Synthesis

1. Obtaining Cry1Fa nucleotide sequence

The amino acid sequence (605 amino acids) of the insecticidal protein mCry1Fa is shown as SEQ ID NO. 3 in the sequence table; the mCry1Fa nucleotide sequence (1818 nucleotides) of the amino acid sequence (605 amino acids) of the insecticidal protein corresponding to the mCry1Fa is shown as SEQ ID NO:1 in the sequence table.

Cry1Fa insecticidal protein amino acid sequence (605 amino acids) is shown as SEQ ID NO. 4 in the sequence table; a Cry1Fa nucleotide sequence (1818 nucleotides) which encodes an amino acid sequence (605 amino acids) corresponding to the Cry1Fa insecticidal protein and is shown as SEQ ID NO:2 in a sequence table.

2. Synthesis of the Cry1Fa nucleotide sequence

The mCry1Fa nucleotide sequence (shown as SEQ ID NO:1 in the sequence table) and the Cry1Fa nucleotide sequence (shown as SEQ ID NO:3 in the sequence table) are synthesized by Nanjing Kinsley biotechnology company; the 5 'end of the synthesized mCry1Fa nucleotide sequence is also connected with an NcoI enzyme cutting site, and the 3' end of the mCry1Fa nucleotide sequence is also connected with an EcoRI enzyme cutting site; the 5 'end of the synthesized Cry1Fa nucleotide sequence is also connected with an NcoI enzyme cutting site, and the 3' end of the Cry1Fa nucleotide sequence is also connected with an EcoRI enzyme cutting site.

The synthetic nucleotide sequence of mCry1Fa was ligated to cloning vector pEASY-T5(Transgen, Beijing, China, CAT: CT501-01) and the procedures were carried out according to the instructions of pEASY-T5 vector manufactured by Transgen, to obtain recombinant cloning vector LP05-T, whose construction scheme is shown in FIG. 1 (where Kan denotes kanamycin resistance gene; Amp denotes ampicillin resistance gene; pUC origin denotes the sequence of the replication region of plasmid pUC, which can direct the replication of double-stranded DNA; LacZ is the initiation codon of LacZ; mCry1Fa is the nucleotide sequence of mCry1Fa (SEQ ID NO: 1).

The recombinant cloning vector LP05-T was then transformed into E.coli T1 competent cells (Transgen, Beijing, China; Cat. No: CD501) by a heat shock method under the following heat shock conditions: 50. mu.l of E.coli T1 competent cells, 10. mu.l of plasmid DNA (recombinant cloning vector LP05-T), water bath at 42 ℃ for 30 seconds; the ampicillin (100 mg/L) coated LB plates (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L, pH adjusted to 7.5 with NaOH) were grown overnight in a water bath at 37 ℃ for 45 minutes (shaking table at 200 rpm). White colonies were picked and cultured overnight in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, ampicillin 100mg/L, pH 7.5 adjusted with NaOH) at 37 ℃. Extracting the plasmid by an alkaline method: centrifuging the bacterial solution at 12000rpm for 1min, removing supernatant, and suspending the precipitated bacterial solution with 100 μ l ice-precooled solution I (25mM Tris-HCl, 10mM EDTA (ethylene diamine tetraacetic acid), 50mM glucose, pH 8.0); add 150. mu.l of freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)), invert the tube 4 times, mix, and place on ice for 3-5 min; adding 150 μ l ice-cold solution III (4M potassium acetate, 2M acetic acid), mixing well immediately, and standing on ice for 5-10 min; centrifuging at 4 deg.C and 12000rpm for 5min, adding 2 times volume of anhydrous ethanol into the supernatant, mixing, and standing at room temperature for 5 min; centrifuging at 4 deg.C and 12000rpm for 5min, removing supernatant, washing precipitate with 70% ethanol, and air drying; the precipitate was dissolved by adding 30. mu.l of RNase (20. mu.g/ml) in TE (10mM Tris-HCl, 1mM EDTA, pH 8.0); bathing in water at 37 deg.C for 30min to digest RNA; storing at-20 deg.C for use.

After the extracted plasmid is subjected to enzyme digestion identification by NcoI and EcoRI, sequencing verification is carried out on positive clones, and the result shows that the nucleotide sequence of mCry1Fa inserted into the recombinant cloning vector LP05-T is the nucleotide sequence shown by SEQ ID NO. 3 in the sequence table, namely the nucleotide sequence of mCry1Fa is correctly inserted.

According to the method for constructing the recombinant cloning vector LP05-T, the synthesized Cry1Fa nucleotide sequence is connected to a cloning vector pEASY-T5 to obtain the recombinant cloning vector LP05CK-T, wherein Cry1Fa is Cry1Fa nucleotide sequence. The Cry1Fa nucleotide sequence in the recombinant cloning vector LP05CK-T is verified to be correctly inserted by enzyme cutting and sequencing.

Example 2 construction of recombinant expression vector containing Cry1Fa Gene

Restriction enzymes NcoI and EcoRI are used to respectively cut recombinant cloning vector LP05-T and expression vector LP-BB (vector backbone: pCAMBIA3301 (available from CAMBIA organization)), the cut fragment of nucleotide sequence of mCry1Fa is inserted between the NcoI and EcoRI sites of expression vector LP-BB, and conventional enzyme cutting methods are used to construct recombinant expression vector LP-PT05, which is well known to those skilled in the art, and the construction process is shown in FIG. 2 (Kan: kanamycin gene; RB: right border; Ubi: maize Uquitin (Ubiquitin) gene promoter (SEQ ID NO:8), mCry1 Fa: mCry1Fa nucleotide sequence (SEQ ID NO:1), Nos: nopaline synthase terminator (SEQ ID NO:6), Ubi: maize Uquitin (Ubiquitin) gene promoter (SEQ ID NO:8), PAT: coding phosphinothricin acetyltransferase gene (SEQ ID NO:7), 35: cauliflower mosaic virus terminator (MV) from Cauliflower (Cauliflower virus) (SEQ ID NO: 35: MV) is constructed ID NO: 9); LB: left border).

Transforming the recombinant expression vector LP-PT05 into an escherichia coli T1 competent cell by a heat shock method, wherein the heat shock condition is as follows: 50 ul of Escherichia coli T1 competent cells, 10 ul of plasmid DNA (recombinant expression vector LP-PT05), water bath at 42 ℃ for 30 seconds; water bath at 37 ℃ for 1 hour (shaking table at 100 rpm); then, the cells were cultured on LB solid plates (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L, pH adjusted to 7.5 with NaOH) containing 50mg/L Kanamycin (Kanamycin) at 37 ℃ for 12 hours, and white colonies were picked up and cultured overnight at 37 ℃ in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, Kanamycin 50mg/L, pH adjusted to 7.5 with NaOH). The plasmid is extracted by an alkaline method. The extracted plasmid is cut by restriction enzymes NcoI and EcoRI and then identified, and the positive clone is sequenced and identified, the result shows that the nucleotide sequence of the recombinant expression vector LP-PT05 between NcoI and EcoRI sites is the nucleotide sequence shown by SEQ ID NO. 1 in the sequence table, namely the nucleotide sequence of mCry1 Fa.

According to the method for constructing the recombinant expression vector LP-PT05, the Cry1Fa nucleotide sequence cut by the NcoI and EcoRI enzyme-digested recombinant cloning vector LP02-T is inserted into the expression vector LP-BB, so that the recombinant expression vector LP-PT05-CK is obtained. Enzyme digestion and sequencing verify that the Cry1Fa nucleotide sequence is formed between NcoI and EcoRI sites of the recombinant expression vector LP-PT 05-CK.

EXAMPLE 3 transformation of Agrobacterium with recombinant expression vector

The correctly constructed recombinant expression vectors LP-PT05 and LP-PT05-CK are transformed into Agrobacterium LBA4404 (Invitron, Chicago, USA; Cat. No. 18313-015) by a liquid nitrogen method under the following transformation conditions: 100. mu.L Agrobacterium LBA4404, 3. mu.L plasmid DNA (recombinant expression vector); placing in liquid nitrogen for 10 minutes, and carrying out warm water bath at 37 ℃ for 10 minutes; inoculating the transformed Agrobacterium LBA4404 in an LB test tube, culturing at 28 ℃ and 200rpm for 2 hours, smearing on an LB plate containing 50mg/L Rifampicin (Rifampicin) and 50mg/L Kanamycin (Kanamycin) until a positive monoclonal is grown, picking out the monoclonal for culturing and extracting the plasmid, carrying out enzyme digestion verification after restriction enzymes NotI and SalI are used for carrying out enzyme digestion on recombinant expression vectors LP-PT05 and LP-PT05-CK, and indicating that the structures of the recombinant expression vectors LP-PT05 and LP-PT05-CK are completely correct.

The conversion comprises the following specific steps:

1) preparation of maize immature embryos

The maize inbred line AX808 in a company is planted in a field or a greenhouse, and maize 8-10 days (summer) to 10-13 days (autumn) after artificial pollination is taken as a source of immature embryos.

2) Preparation of Agrobacterium

(1) Marking the transformed and identified agrobacterium tumefaciens glycerol on a YEP solid culture medium added with 100mg/L kan and 12 mg/L tet, and culturing in dark at 28 ℃ for 2-3 days;

(2) adding 1ml of infection culture medium into a sterilized 2ml centrifugal tube, putting the agrobacterium of the step 1 into the infection culture medium, and fully scattering and uniformly mixing the agrobacterium with a pipette gun;

(3) and (3) taking 1 sterilized 2ml centrifuge tube, and adjusting the concentration of the bacterial liquid by using an infection culture medium to ensure that the OD 660 is 0.5-0.7.

3) Co-culture of young maize embryos and agrobacterium

(1) Removing the infection culture medium in the immature embryo centrifuge tube, and adding 1.5ml of fresh infection culture medium to clean the embryo once;

(2) removing the infection culture medium, and adding the adjusted agrobacterium liquid;

(3) oscillating at the maximum rotating speed for 30s, and standing at room temperature for 5 min;

(4) pouring the embryos onto a co-culture medium, and blotting the liquid;

(5) placing the embryo with the plane upward and the shield surface upward;

(6) the embryos are cultured in the dark at 22 ℃ for 2-3 days.

4) Induction and selection of calli

(1) Transferring the co-cultured embryo to an induced callus culture medium, and performing dark culture in an incubator at 28 ℃ for 7-10 days;

(2) transferring the induced callus to a screening culture medium for screening culture, wherein the screening pressure is 5.0 mM glyphosate, and dark culture is carried out for 2-3 weeks at 28 ℃;

(3) taking the callus survived in the first screening to carry out the second screening, wherein the screening pressure is 2.0 mM;

5) regeneration and culture of transformed strains

(1) Placing the screened embryogenic callus on a pre-differentiation culture medium, and performing dark culture at 28 deg.C for 10-14 days;

(2) taking embryo healing wound on a differentiation culture medium, and performing light culture at 28 ℃ for 10-14 days until the seedling is differentiated;

(3) transferring the differentiated seedling to a rooting culture medium, and performing light culture at 28 ℃ until the root is completely developed;

(4) transplanting the well-grown seedlings into a greenhouse matrix.

And harvesting the transgenic plants after the transgenic plants blossom and fruit. The harvested seeds are sowed in a greenhouse, and when the plants grow to 4-6 leaf stages, expression analysis and detection are carried out by adopting a PCR technology.

2. Maize plants transformed with the Cry1Fa gene were verified by ordinary PCR using 2 × EasyTaq PCR Supermix (China, Beijing, Cat: AS111-11) from Hokken corporation.

The primers for PCR were:

Cry1Fa-393F(SEQ ID NO:10)：cgtcatcccaacttacaagg

Cry1Fa-930R(SEQ ID NO:11)：CATGATTCAGCACATGGGAG

fragment size: 510bp

The conditions for the PCR reaction were: 30 cycles, each cycle being 95 ℃ 30 ', 58 ℃ 30 ', 72 ℃ 40 '

3) Verification of Cry1Fa Gene transferred maize plants by qRT-PCR

About 100mg of leaves of a maize Plant with an mCry1Fa transferred nucleotide sequence and a maize Plant with a Cry1Fa transferred nucleotide sequence are taken as samples respectively, Genomic DNA of the leaves is extracted by an easy pure Plant Genomic DNA Kit (containing RNase A) of Transgen (Transgen, Beijing, China, Cat: EE111-01), and the copy number of the mCry1Fa gene is detected by a TransStart Green fluorescent quantitative PCR method. Meanwhile, wild corn plants are used as a control, and detection and analysis are carried out according to the method. The experiment was repeated 3 times and the average was taken.

The specific method for detecting the copy number of the Cry1Fa gene is as follows:

step 11, respectively taking 100mg of leaves of the corn plant with the mCry1Fa nucleotide sequence and the wild corn plant, respectively grinding the leaves into homogenate in a mortar by using liquid nitrogen, and taking 3 samples for repetition;

step 12, extracting the Genomic DNA of the sample by using an easy pure Plant Genomic DNA Kit (containing RNase A) of Transgen (Transgen, Beijing, China, Cat: EE111-01), and referring to the product specification of the specific method;

step 13, measuring the genomic DNA concentration of the sample by using NanoDrop 2000(Thermo Scientific);

step 14, adjusting the genomic DNA concentration of the sample to the same concentration value, wherein the concentration value range is 80-100 ng/mu l;

step 15, identifying the copy number of the sample by adopting a TransStart Green fluorescent quantitative PCR method, taking the sample with known copy number after identification as a standard substance, taking the sample of a wild corn plant as a control, repeating each sample for 3 times, and taking the average value; the fluorescent quantitative PCR primer and the probe sequence are respectively as follows:

the following primers were used to detect mCry1Fa and Cry1Fa nucleotide sequences:

primer 1(CF 5): CATTCGCGTACACCATTGTC is shown as SEQ ID NO:12 in the sequence list;

primer 2(CR 5): ACCAGCAAAGATCCGTTCAC is shown as SEQ ID NO. 13 in the sequence list;

the following primers were used to detect the 18s nucleotide sequence for internal reference leveling

18srRNA-F(SEQ ID NO:14)：CCATCCCTCCGTAGTTAGCTTCT

18srRNA-R(SEQ IDNO:15)：CCTGTCGGCCAAGGCTATATAC

The PCR reaction system is as follows:

the PCR reaction conditions are as follows:

step (ii) of

Returning to the step 2, circulating for 40 times

Data were analyzed using SDS2.3 software (Applied Biosystems).

As shown in FIG. 3, the nucleotide sequences of mCry1Fa and Cry1Fa are integrated into the chromosome group of the detected corn plants, and the corn plants with the mCry1Fa nucleotide sequences are transformed to obtain transgenic corn plants containing single copies of mCry1Fa and Cry1Fa genes.

Example 4 insecticidal protein detection of transgenic maize plants

1. Content detection of insecticidal protein (Cry1Fa protein) of transgenic corn plants

The solutions involved in this experiment were as follows:

extracting a buffer solution: 8g/L NaCl, 0.2g/L KH2PO4, 2.9g/L Na2HPO 4.12H 2O, 0.2g/L KCl, 5.5ml/L Tween 20(Tween-20), pH 7.4;

wash buffer PBST: 8g/L NaCl, 0.2g/L KH2PO4, 2.9g/L Na2HPO 4.12H 2O, 0.2g/LKCl, 0.5ml/L Tween 20(Tween-20), pH 7.4;

stopping liquid: 1M HCl.

Taking fresh leaves of 3mg of single-copy corn plant with the mCry1Fa nucleotide sequence and single-copy corn plant with the Cry1Fa nucleotide sequence as samples, grinding by liquid nitrogen, adding 800 mu l of the extraction buffer solution, centrifuging for 10min at the rotating speed of 4000rpm, taking supernatant, diluting by 40 times by using the extraction buffer solution, and taking 80 mu l of diluted supernatant for ELISA detection. The proportion of the insecticidal protein (Cry1Fa protein) in the sample to the fresh weight of the leaves is detected and analyzed by an ELISA (enzyme-linked immunosorbent assay) kit (ENVIRONLOGIX, Cry1F kit, AP016), and the specific method refers to the product specification thereof.

Meanwhile, wild corn plants and corn plants which are identified as non-transgenic through fluorescent quantitative PCR are used as controls, and detection and analysis are carried out according to the method. 3 strains (F1, F2 and F3) which are transferred into the mCry1Fa nucleotide sequence, 3 strains (F4, F5 and F6) which are transferred into the Cry1Fa nucleotide sequence, 1 strain which is identified as non-transgenic (NGM) and 1 strain which is wild type (CK) through fluorescence quantitative PCR; 3 strains from each line were selected for testing, each repeated 6 times.

The experimental results for the insecticidal protein (Cry1Fa protein) content of the transgenic corn plants are shown in table 1. The ratio (ng/g) of the average expression quantity of the insecticidal protein (Cry1Fa protein) in the fresh leaves of the corn plant with the mCry1Fa nucleotide sequence and the corn plant with the Cry1Fa nucleotide sequence to the fresh weight of the leaves is respectively determined to be 4642.20 and 4555.42, and the result shows that the Cry1Fa protein obtains higher expression quantity and stability in the corn.

TABLE 1 average Cry1Fa protein expression measurements of transgenic maize plants

The steps of in vitro expression and purification of the mCry1Fa protein are as follows:

1. artificially synthesized double-stranded DNA molecule shown as SEQ ID NO 1 in sequence table

2. And (3) connecting the double-stranded DNA molecule synthesized in the step (1) with a prokaryotic expression vector pEASY-E1 to obtain a recombinant plasmid pEASY-mCry1 Fa. The recombinant plasmid pEASY-mCry1Fa was sequenced. The sequencing result shows that the recombinant plasmid pEASY-mCry1Fa contains a DNA molecule shown by SEQ ID NO. 1 in a sequence table, and expresses mCry1Fa protein shown by SEQ ID NO. 3 in the sequence table.

3. The recombinant plasmid pEASY-mCry1Fa is introduced into Escherichia coli transetta to obtain a recombinant bacterium, and the recombinant bacterium is named as transetta-mCry1 Fa.

4. A single clone of transetta-mCry1Fa was inoculated into 100mL of LB liquid medium (containing 50. mu.g/mL of ampicillin), and cultured at 37 ℃ and 200rpm for 12 hours with shaking to obtain a culture broth.

5. Inoculating the cultured bacterial liquid into 50mL LB liquid medium (containing 50. mu.g/mL ampicillin) at a volume ratio of 1:100, performing shake culture at 37 deg.C and 200rpm until OD600nm value is 0.6, adding IPTG to a concentration of 1mM, performing shake culture at 28 deg.C and 220rpm for 4h, centrifuging at 4 deg.C and 10000rpm for 10min, and collecting thallus precipitate.

6. Collecting thallus precipitate, adding 100mL Tris-HCl buffer solution with pH of 8.0 and 100mM, carrying out ultrasonication (ultrasonic power 600W, cycle program: crushing for 4s, stopping for 6s, totally 20min), centrifuging at 4 deg.C and 10000rpm for 10min, and collecting supernatant A.

7. Taking the supernatant A, centrifuging at 4 ℃ and 12000rpm for 10min, and collecting the supernatant B.

8. The supernatant b was purified using a nickel column manufactured by GE (the specific steps of purification refer to the specifications of the nickel column), and then mCry1Fa protein was quantified using a protein quantification kit manufactured by seimer feishel.

According to the method, the step 1 of artificially synthesizing the double-stranded DNA molecule shown as SEQ ID NO. 1 in the sequence table is replaced by the step of artificially synthesizing the double-stranded DNA molecule shown as SEQ ID NO. 2 in the sequence table, and other steps are not changed, so that the Cry1Fa protein is obtained.

9. Biological assay method for spodoptera frugiperda and cutworm

The activity of mcry1Fa protein and cry1Fa protein were compared according to the bioassay method reported by jin Xue (2008), and the insecticidal activity of the two proteins (mcry1Fa protein and cry1Fa protein) against four lepidopteran insects (spodoptera frugiperda and agrotis microti) was determined separately, and the results are shown in Table 2.

10. Detection of insect-resistant Effect of transgenic maize plants

The corn plant with the transferred Cry1Fa gene sequence, the corn plant with the transferred optimized mCry1Fa gene sequence, the wild corn plant and the corn plant identified as non-transgenic by fluorescence quantitative PCR are respectively used for detecting the insect-resistant effect of spodoptera frugiperda and cutworm.

(1) Spodoptera frugiperda: fresh leaves of a corn plant with an optimized mCry1Fa gene sequence, a corn plant with a known Cry1Fa gene sequence, a wild corn plant and a corn plant identified as a non-transgenic plant by fluorescence quantitative PCR are respectively taken, washed clean in sterile water, water on the leaves is sucked by gauze, strips are cut at the same time, 3 cut strips are taken and put on filter paper at the bottom of a circular plastic culture dish, the filter paper is wetted by distilled water, 10 artificially fed spodoptera frugiperda and black cutworm (hatched larva) are put in each culture dish, the death condition of the larva is counted after the culture dish is covered, the larva is placed for 3-5 days under the conditions of the temperature of 26-28 ℃, the relative humidity of 70-80% and the light period (light/dark) of 16: 8, and the average death rate of the ostrinia nubilalis in each sample is calculated. The results are shown in Table 3 after transferring 4 strains (F4, F5 and F6) with optimized Cry1Fa gene sequence and transferring 3 strains (F1, F2 and F3) with known optimized Cry1Fa gene sequence.

10. The efficiency of transgenic Cry1Fa maize plants against black cutworm and spodoptera frugiperda was determined by damage to maize:

1) plant treatment:

stage V3 controls and transgenic Cry1Fa seedlings, insects: two-instar larva of black cutworm

Six replicates of each material were tested, 6 seedlings per replicate, one larva per seedling. Comparison: 6 replicates of 6 seedlings, 3 larvae per seedling.

2) The process comprises the following steps:

each replicate contained 6 seeds sown into a transparent rectangular tray (15cmX25cm) placed within the mesh to limit the range of larvae movement, 6 larvae per replicate infected. The discs were placed in 25% and 75% humidity conditions and infected for 72 hours to assess infection. The result is shown in Table 4, the resistance of mCry1Fa to the living body bioassay of cutworm and Spodoptera frugiperda is far higher than that of Cry1Fa, and the mCry1Fa gene disclosed by the invention has a better insect-resistant effect and better plant protection.

3) Evaluation of

Plants-classified undamaged, damaged or cut under cotyledons. Each replicate was graded: 1 (undamaged or having some nicking by the trade) to 9 (whole plants were cut out and more than 80% of the tissue was eaten) with the results shown in table 4 and figure 4.

4) Technical effects

After the mCry1Fa gene is transferred into a plant, the obtained transgenic plant of the mCry1Fa protein with high expression is superior to the transgenic plant of Cry1Fa protein with high expression in the resistance of the black cutworm.

TABLE 2

TABLE 3 in vitro lethality of mCry1Fa plants

Note: the killing efficiency of the mCry1Fa to black cutworms and spodoptera frugiperda is far higher than that of Cry1Fa, and the gene of the mCry1Fa has better insecticidal effect

TABLE 4 in vivo results of mCry1Fa on Tiger

Note: the resistance of mCry1Fa to living body bioassay of agrotis ypsilon is far higher than that of Cry1Fa, and the mCry1Fa gene disclosed by the invention has a better insect-resistant effect and better plant protection effect

Although the present application has been described in detail with respect to the general description and the specific examples, it will be apparent to those skilled in the art that certain changes and modifications may be made based on the present application. Accordingly, such modifications and improvements are intended to be within the scope of this invention as claimed.

Sequence listing

SEQ ID NO 1 mCry1Fa sequence (1818bp)

atggagaacaacatacagaatcagtgcattccctttaactgcctcaacaatcctgaagtagagattctcaacgaagagaggtcgactggcagattgccg ttagacatctccctgtcccttacacgtttcctgttgtctgagtttgttccaggtgtgggagttgcgtttggcctcttcgacctcatctggggcttcatcactcca tctgattggagcctctttcttctccagattgaacagttgattgaacaaaggattgagaccttggaaaggaatcgggccatcactacccttcgtggcttagca gacagctatgagatctacattgaagcactaagagagtgggaagccaatcctaacaatgcccaactgagagaagatgtgcgtatacgctttgctaacac agatgatgctttgatcacagccatcaacaacttcacccttaccagcttcgagatccctcttctctcggtctatgttcaagctgctaacctgcacttgtcacta ctgcgcgacgctgtgtcgtttgggcaaggttggggactggacatagctactgtcaacaatcactacaacagactcatcaatctgattcatcgatacacga aacattgtttggatacctacaatcagggattggagaacctgagaggtactaacactcgccaatgggccaggttcaatcagttcaggagagaccttacac ttactgtgttagacatagttgctctctttccgaactacgatgttcgtacctatccgattcaaacgtcatcccaacttacaagggagatctacaccagttcagtc attgaagactctccagtttctgcgaacatacccaatggtttcaacagggctgagtttggagtcagaccaccccatctcatggacttcatgaactctttgtttg tgactgcagagactgttagatcccaaactgtgtggggaggacacttagttagctcacgcaacacggctggcaatcgtatcaactttcctagttacggggt cttcaatcccgggggcgccatctggattgcagatgaagatccacgtcctttctatcggaccttgtcagatcctgtcttcgtccgaggaggctttggcaatc ctcactatgtactcggtcttaggggagtggcctttcaacaaactggtacgaatcacacccgcacattcaggaactccgggaccattgactctctagatga gataccacctcaagacaacagcggcgcaccttggaatgactactcccatgtgctgaatcatgttacctttgtgcgctggccaggtgagatctcaggttcc gactcatggagagcaccaatgttctcttggacgcatcgtagcgctacccccacaaacaccattgatccagagagaatcactcagattcccttggtgaag gcacacacacttcagtcaggaactacagttgtaagagggccggggttcacgggaggagacattcttcgacgcactagtggaggaccattcgcgtaca ccattgtcaacatcaatgggcaacttccccaaaggtatcgtgccaggatacgctatgcctctactaccaatctaagaatctacgttacggttgcaggtgaa cggatctttgctggtcagttcaacaagacaatggataccggtgatccacttacattccaatctttctcctacgccactatcaacaccgcgttcacctttccaa tgagccagagcagtttcacagtaggtgctgataccttcagttcaggcaacgaagtgtacattgacaggtttgagttgattccagttactgccacactcgag taa

SEQ ID NO 2 Cry1Fa gene sequence

atggagaacaacatacagaatcagtgcgtcccctacaactgcctcaacaatcctgaagtagagattctcaacgaagagaggtcgactggcagattgcc gttagacatctccctgtcccttacacgtttcctgttgtctgagtttgttccaggtgtgggagttgcgtttggcctcttcgacctcatctggggcttcatcactcc atctgattggagcctctttcttctccagattgaacagttgattgaacaaaggattgagaccttggaaaggaatcgggccatcactacccttcgtggcttagc agacagctatgagatctacattgaagcactaagagagtgggaagccaatcctaacaatgcccaactgagagaagatgtgcgtatacgctttgctaaca cagatgatgctttgatcacagccatcaacaacttcacccttaccagcttcgagatccctcttctctcggtctatgttcaagctgctaacctgcacttgtcact actgcgcgacgctgtgtcgtttgggcaaggttggggactggacatagctactgtcaacaatcactacaacagactcatcaatctgattcatcgatacacg aaacattgtttggatacctacaatcagggattggagaacctgagaggtactaacactcgccaatgggccaggttcaatcagttcaggagagaccttaca cttactgtgttagacatagttgctctctttccgaactacgatgttcgtacctatccgattcaaacgtcatcccaacttacaagggagatctacaccagttcagt cattgaagactctccagtttctgcgaacatacccaatggtttcaacagggctgagtttggagtcagaccaccccatctcatggacttcatgaactctttgttt gtgactgcagagactgttagatcccaaactgtgtggggaggacacttagttagctcacgcaacacggctggcaatcgtatcaactttcctagttacggg gtcttcaatcccgggggcgccatctggattgcagatgaagatccacgtcctttctatcggaccttgtcagatcctgtcttcgtccgaggaggctttggcaa tcctcactatgtactcggtcttaggggagtggcctttcaacaaactggtacgaatcacacccgcacattcaggaactccgggaccattgactctctagat gagataccacctcaagacaacagcggcgcaccttggaatgactactcccatgtgctgaatcatgttacctttgtgcgctggccaggtgagatctcaggtt ccgactcatggagagcaccaatgttctcttggacgcatcgtagcgctacccccacaaacaccattgatccagagagaatcactcagattcccttggtga aggcacacacacttcagtcaggaactacagttgtaagagggccggggttcacgggaggagacattcttcgacgcactagtggaggaccattcgcgta caccattgtcaacatcaatgggcaacttccccaaaggtatcgtgccaggatacgctatgcctctactaccaatctaagaatctacgttacggttgcaggtg aacggatctttgctggtcagttcaacaagacaatggataccggtgatccacttacattccaatctttctcctacgccactatcaacaccgcgttcacctttcc aatgagccagagcagtttcacagtaggtgctgataccttcagttcaggcaacgaagtgtacattgacaggtttgagttgattccagttactgccacactcg agtaa

3 mCry1Fa protein sequence of SEQ ID NO

MENNIQNQCIPFNCLNNPEVEILNEERSTGRLPLDISLSLTRFLLSEFVPGVGVAFGLFDLIWGFITPSDWSLFLLQIEQLIEQRIETLERNRAITTLRGLADSYEIYIEALREWEANPNNAQLREDVRIRFA NTDDALITAINNFTLTSFEIPLLSVYVQAANLHLSLLRDAVSFGQGWGLDIATVNNHYNRLINLIH RYTKHCLDTYNQGLENLRGTNTRQWARFNQFRRDLTLTVLDIVALFPNYDVRTYPIQTSSQLTR EIYTSSVIEDSPVSANIPNGFNRAEFGVRPPHLMDFMNSLFVTAETVRSQTVWGGHLVSSRNTA GNRINFPSYGVFNPGGAIWIADEDPRPFYRTLSDPVFVRGGFGNPHYVLGLRGVAFQQTGTNHT RTFRNSGTIDSLDEIPPQDNSGAPWNDYSHVLNHVTFVRWPGEISGSDSWRAPMFSWTHRSATP TNTIDPERITQIPLVKAHTLQSGTTVVRGPGFTGGDILRRTSGGPFAYTIVNINGQLPQRYRARIRY ASTTNLRIYVTVAGERIFAGQFNKTMDTGDPLTFQSFSYATINTAFTFPMSQSSFTVGADTFSSGNEVYIDRFELIPVTATLE

4 Cry1Fa protein sequence of SEQ ID NO

MENNIQNQCVPYNCLNNPEVEILNEERSTGRLPLDISLSLTRFLLSEFVPGVGVAFGLFDLIWGFI TPSDWSLFLLQIEQLIEQRIETLERNRAITTLRGLADSYEIYIEALREWEANPNNAQLREDVRIRF ANTDDALITAINNFTLTSFEIPLLSVYVQAANLHLSLLRDAVSFGQGWGLDIATVNNHYNRLINLI HRYTKHCLDTYNQGLENLRGTNTRQWARFNQFRRDLTLTVLDIVALFPNYDVRTYPIQTSSQLT REIYTSSVIEDSPVSANIPNGFNRAEFGVRPPHLMDFMNSLFVTAETVRSQTVWGGHLVSSRNTA GNRINFPSYGVFNPGGAIWIADEDPRPFYRTLSDPVFVRGGFGNPHYVLGLRGVAFQQTGTNHT RTFRNSGTIDSLDEIPPQDNSGAPWNDYSHVLNHVTFVRWPGEISGSDSWRAPMFSWTHRSATP TNTIDPERITQIPLVKAHTLQSGTTVVRGPGFTGGDILRRTSGGPFAYTIVNINGQLPQRYRARIRY ASTTNLRIYVTVAGERIFAGQFNKTMDTGDPLTFQSFSYATINTAFTFPMSQSSFTVGADTFSSGNEVYIDRFELIPVTATLE

SEQ ID NO 5 p35s nt sequence

Ccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggccatcgttgaa gatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtg gattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagagg acacgctgacaagctgactctagcagatctaccgtcttcggtacgcgctcactccgccctctgcctttgttactgccacgtttctctgaatgctctcttgtgt ggtgattgctgagagtggtttagctggatctagaattacactctgaaatcgtgttctgcctgtgctgattacttgccgtcctttgtagcagcaaaatataggg acatggtagtacgaaacgaagatagaacctacacagcaatacgagaaatgtgtaatttggtgcttagcggtatttatttaagcacatgttggtgttatagg gcacttggattcagaagtttgctgttaatttaggcacaggcttcatactacatgggtcaatagtatagggattcatattataggcgatactataataatttgttc gtctgcagagcttattatttgccaaaattagatattcctattctgtttttgtttgtgtgctgttaaattgttaacgcctgaaggaataaatataaatgacgaaatttt gatgtttatctctgctcctttattgtgaccataagtcaagatcagatgcacttgttttaaatattgttgtctgaagaaataagtactgacagtattttgatgcattg atctgcttgtttgttgtaacaaaatttaaaaataaagagtttcctttttgttgctctccttacctcctgatggtatctagtatctaccaactgacactatattgcttct ctttacatacgtatcttgctcgatgccttctccctagtgttgaccagtgttactcacatagtctttgctcatttcattgtaatgcagataccaagcggcc

SEQ ID NO 6 Nos nt sequence

Gatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataa ttaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaa ctaggataaattatcgcgcgcggtgtcatctatgttactagatc

7 cPAT nt sequence of SEQ ID NO

atgtctccggagaggagaccagttgagattaggccagctacagcagctgatatggccgcggtttgtgatatcgttaaccattacattgagacgtctacag tgaactttaggacagagccacaaacaccacaagagtggattgatgatctagagaggttgcaagatagatacccttggttggttgctgaggttgagggtg ttgtggctggtattgcttacgctgggccctggaaggctaggaacgcttacgattggacagttgagagtactgtttacgtgtcacataggcatcaaaggttg ggcctaggatccacattgtacacacatttgcttaagtctatggaggcgcaaggttttaagtctgtggttgctgttataggccttccaaacgatccatctgtta ggttgcatgaggctttgggatacacagcccggggtacattgcgcgcagctggatacaagcatggtggatggcatgatgttggtttttggcaaagggatt ttgagttgccagctcctccaaggccagttaggccagttacccagatctga

8 pUbi nt sequence of SEQ ID NO

ctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttgtttg aagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaatcatataaatgaac agttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacct atataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaatta agaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaa aaactaaggaaacatttttcttgtttcgagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtc gcgtcgggccaagcgaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcg gcatccagaaattgcgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcaccggcagctacgggggattc ctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacac acacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctctaccttctctagatc ggcgttccggtgcatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttcgtacacggat gcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttccgcagacgggatcgatttc atgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttg tgatgatgtggtctggttgggcggtcgttctagatcggagtagatttctgtttcaaactacctggtggatttattaattttggatctgtatgtgtgtgccatacat attcatagttacgaattgaagatgatggatggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgtt cgcttggttgtgatgatgtggtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaact gtatgtgtgtgtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatataca tgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatg gcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttactt ctgcag

935 s Nt sequence of SEQ ID NO

Ctgaaatcaccagtctctctctacaaatctatctctctctataataatgtgtgagtagttcccagataagggaattagggttcttatagggtttcgctcatgtgt tgagcatataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtgg

SEQ ID NO:10 Cry1Fa-393F

cgtcatcccaacttacaagg

SEQ ID NO:11 Cry1Fa-930R

CATGATTCAGCACATGGGAG

SEQ ID NO:12 CF5

CATTCGCGTACACCATTGTC

SEQ ID NO:13 CR5

ACCAGCAAAGATCCGTTCAC

SEQ ID NO:14 18srRNA-F

CCATCCCTCCGTAGTTAGCTTCT

SEQ ID NO:15 18srRNA-R

CCTGTCGGCCAAGGCTATATAC 。

Sequence listing

<110> Longping Biotechnology (Hainan) Co., Ltd

<120> plant insect-resistant protein and coding gene and application thereof

<130>20200515

<160>15

<170>SIPOSequenceListing 1.0

<210>1

<211>1818

<212>DNA

<213>Bacillus thuringiensis

<400>1

atggagaaca acatacagaa tcagtgcatt ccctttaact gcctcaacaa tcctgaagta 60

gagattctca acgaagagag gtcgactggc agattgccgt tagacatctc cctgtccctt 120

acacgtttcc tgttgtctga gtttgttcca ggtgtgggag ttgcgtttgg cctcttcgac 180

ctcatctggg gcttcatcac tccatctgat tggagcctct ttcttctcca gattgaacag 240

ttgattgaac aaaggattga gaccttggaa aggaatcggg ccatcactac ccttcgtggc 300

ttagcagaca gctatgagat ctacattgaa gcactaagag agtgggaagc caatcctaac 360

aatgcccaac tgagagaaga tgtgcgtata cgctttgcta acacagatga tgctttgatc 420

acagccatca acaacttcac ccttaccagc ttcgagatcc ctcttctctc ggtctatgtt 480

caagctgcta acctgcactt gtcactactg cgcgacgctg tgtcgtttgg gcaaggttgg 540

ggactggaca tagctactgt caacaatcac tacaacagac tcatcaatct gattcatcga 600

tacacgaaac attgtttgga tacctacaat cagggattgg agaacctgag aggtactaac 660

actcgccaat gggccaggtt caatcagttc aggagagacc ttacacttac tgtgttagac 720

atagttgctc tctttccgaa ctacgatgtt cgtacctatc cgattcaaac gtcatcccaa 780

cttacaaggg agatctacac cagttcagtc attgaagact ctccagtttc tgcgaacata 840

cccaatggtt tcaacagggc tgagtttgga gtcagaccac cccatctcat ggacttcatg 900

aactctttgt ttgtgactgc agagactgtt agatcccaaa ctgtgtgggg aggacactta 960

gttagctcac gcaacacggc tggcaatcgt atcaactttc ctagttacgg ggtcttcaat 1020

cccgggggcg ccatctggat tgcagatgaa gatccacgtc ctttctatcg gaccttgtca 1080

gatcctgtct tcgtccgagg aggctttggc aatcctcact atgtactcgg tcttagggga 1140

gtggcctttc aacaaactgg tacgaatcac acccgcacat tcaggaactc cgggaccatt 1200

gactctctag atgagatacc acctcaagac aacagcggcg caccttggaa tgactactcc 1260

catgtgctga atcatgttac ctttgtgcgc tggccaggtg agatctcagg ttccgactca 1320

tggagagcac caatgttctc ttggacgcat cgtagcgcta cccccacaaa caccattgat 1380

ccagagagaa tcactcagat tcccttggtg aaggcacaca cacttcagtc aggaactaca 1440

gttgtaagag ggccggggtt cacgggagga gacattcttc gacgcactag tggaggacca 1500

ttcgcgtaca ccattgtcaa catcaatggg caacttcccc aaaggtatcg tgccaggata 1560

cgctatgcct ctactaccaa tctaagaatc tacgttacgg ttgcaggtga acggatcttt 1620

gctggtcagt tcaacaagac aatggatacc ggtgatccac ttacattcca atctttctcc 1680

tacgccacta tcaacaccgc gttcaccttt ccaatgagcc agagcagttt cacagtaggt 1740

gctgatacct tcagttcagg caacgaagtg tacattgaca ggtttgagtt gattccagtt 1800

actgccacac tcgagtaa 1818

<210>2

<211>1818

<212>DNA

<213>Bacillus thuringiensis

<400>2

atggagaaca acatacagaa tcagtgcgtc ccctacaact gcctcaacaa tcctgaagta 60

gagattctca acgaagagag gtcgactggc agattgccgt tagacatctc cctgtccctt 120

acacgtttcc tgttgtctga gtttgttcca ggtgtgggag ttgcgtttgg cctcttcgac 180

ctcatctggg gcttcatcac tccatctgat tggagcctct ttcttctcca gattgaacag 240

ttgattgaac aaaggattga gaccttggaa aggaatcggg ccatcactac ccttcgtggc 300

ttagcagaca gctatgagat ctacattgaa gcactaagag agtgggaagc caatcctaac 360

aatgcccaac tgagagaaga tgtgcgtata cgctttgcta acacagatga tgctttgatc 420

acagccatca acaacttcac ccttaccagc ttcgagatcc ctcttctctc ggtctatgtt 480

caagctgcta acctgcactt gtcactactg cgcgacgctg tgtcgtttgg gcaaggttgg 540

ggactggaca tagctactgt caacaatcac tacaacagac tcatcaatct gattcatcga 600

tacacgaaac attgtttgga tacctacaat cagggattgg agaacctgag aggtactaac 660

actcgccaat gggccaggtt caatcagttc aggagagacc ttacacttac tgtgttagac 720

atagttgctc tctttccgaa ctacgatgtt cgtacctatc cgattcaaac gtcatcccaa 780

cttacaaggg agatctacac cagttcagtc attgaagact ctccagtttc tgcgaacata 840

cccaatggtt tcaacagggc tgagtttgga gtcagaccac cccatctcat ggacttcatg 900

aactctttgt ttgtgactgc agagactgtt agatcccaaa ctgtgtgggg aggacactta 960

gttagctcac gcaacacggc tggcaatcgt atcaactttc ctagttacgg ggtcttcaat 1020

cccgggggcg ccatctggat tgcagatgaa gatccacgtc ctttctatcg gaccttgtca 1080

gatcctgtct tcgtccgagg aggctttggc aatcctcact atgtactcgg tcttagggga 1140

gtggcctttc aacaaactgg tacgaatcac acccgcacat tcaggaactc cgggaccatt 1200

gactctctag atgagatacc acctcaagac aacagcggcg caccttggaa tgactactcc 1260

catgtgctga atcatgttac ctttgtgcgc tggccaggtg agatctcagg ttccgactca 1320

tggagagcac caatgttctc ttggacgcat cgtagcgcta cccccacaaa caccattgat 1380

ccagagagaa tcactcagat tcccttggtg aaggcacaca cacttcagtc aggaactaca 1440

gttgtaagag ggccggggtt cacgggagga gacattcttc gacgcactag tggaggacca 1500

ttcgcgtaca ccattgtcaa catcaatggg caacttcccc aaaggtatcg tgccaggata 1560

cgctatgcct ctactaccaa tctaagaatc tacgttacgg ttgcaggtga acggatcttt 1620

gctggtcagt tcaacaagac aatggatacc ggtgatccac ttacattcca atctttctcc 1680

tacgccacta tcaacaccgc gttcaccttt ccaatgagcc agagcagttt cacagtaggt 1740

gctgatacct tcagttcagg caacgaagtg tacattgaca ggtttgagtt gattccagtt 1800

actgccacac tcgagtaa 1818

<210>3

<211>605

<212>PRT

<213>Bacillus thuringiensis

<400>3

Met Glu Asn Asn Ile Gln Asn Gln Cys Ile Pro Phe Asn Cys Leu Asn

1 5 10 15

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

20 25 30

Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe

35 40 45

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

50 55 60

Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln

65 70 75 80

Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr

85 90 95

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

100105 110

Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val

115 120 125

Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn

130 135 140

Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val

145 150 155 160

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

165 170 175

Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn

180 185 190

Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr

195 200 205

Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp

210 215 220

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

225 230 235 240

Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln

245 250 255

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

260 265270

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

275 280 285

Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe

290 295 300

Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu

305 310 315 320

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

325 330 335

Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro

340 345 350

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

355 360 365

Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln

370 375 380

Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile

385 390 395 400

Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp

405 410 415

Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro

420 425430

Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp

435 440 445

Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile

450 455 460

Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr

465 470 475 480

Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr

485 490 495

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

500 505 510

Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu

515 520 525

Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe

530 535 540

Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser

545 550 555 560

Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser

565 570 575

Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile

580 585590

Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu

595 600 605

<210>4

<211>605

<212>PRT

<213>Bacillus thuringiensis

<400>4

Met Glu Asn Asn Ile Gln Asn Gln Cys Val Pro Tyr Asn Cys Leu Asn

1 5 10 15

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

20 25 30

Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Phe Leu Leu Ser Glu Phe

35 40 45

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

50 55 60

Phe Ile Thr Pro Ser Asp Trp Ser Leu Phe Leu Leu Gln Ile Glu Gln

65 70 75 80

Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Arg Asn Arg Ala Ile Thr

85 90 95

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

100 105 110

Arg Glu Trp Glu Ala Asn Pro Asn Asn Ala Gln Leu Arg Glu Asp Val

115 120 125

Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Leu Ile Thr Ala Ile Asn

130 135 140

Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Leu Leu Ser Val Tyr Val

145 150 155 160

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

165 170 175

Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Val Asn Asn His Tyr Asn

180 185 190

Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Lys His Cys Leu Asp Thr

195 200 205

Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Thr Asn Thr Arg Gln Trp

210 215 220

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

225 230 235 240

Ile Val Ala Leu Phe Pro Asn Tyr Asp Val Arg Thr Tyr Pro Ile Gln

245 250 255

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

260 265 270

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

275 280 285

Phe Gly Val Arg Pro Pro His Leu Met Asp Phe Met Asn Ser Leu Phe

290 295 300

Val Thr Ala Glu Thr Val Arg Ser Gln Thr Val Trp Gly Gly His Leu

305 310 315 320

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

325 330 335

Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Ala Asp Glu Asp Pro

340 345 350

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

355 360 365

Phe Gly Asn Pro His Tyr Val Leu Gly Leu Arg Gly Val Ala Phe Gln

370 375 380

Gln Thr Gly Thr Asn His Thr Arg Thr Phe Arg Asn Ser Gly Thr Ile

385 390 395 400

Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp Asn Ser Gly Ala Pro Trp

405 410 415

Asn Asp Tyr Ser His Val Leu Asn His Val Thr Phe Val Arg Trp Pro

420 425 430

Gly Glu Ile Ser Gly Ser Asp Ser Trp Arg Ala Pro Met Phe Ser Trp

435 440 445

Thr His Arg Ser Ala Thr Pro Thr Asn Thr Ile Asp Pro Glu Arg Ile

450 455 460

Thr Gln Ile Pro Leu Val Lys Ala His Thr Leu Gln Ser Gly Thr Thr

465 470 475 480

Val Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr

485 490 495

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

500 505 510

Pro Gln Arg Tyr Arg Ala Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu

515 520 525

Arg Ile Tyr Val Thr Val Ala Gly Glu Arg Ile Phe Ala Gly Gln Phe

530 535 540

Asn Lys Thr Met Asp Thr Gly Asp Pro Leu Thr Phe Gln Ser Phe Ser

545 550 555 560

Tyr Ala Thr Ile Asn Thr Ala Phe Thr Phe Pro Met Ser Gln Ser Ser

565 570 575

Phe Thr Val Gly Ala Asp Thr Phe Ser Ser Gly Asn Glu Val Tyr Ile

580 585 590

Asp Arg Phe Glu Leu Ile Pro Val Thr Ala Thr Leu Glu

595 600 605

<210>5

<211>1134

<212>DNA

<213>p35s nt

<400>5

ccattgccca gctatctgtc actttattgt gaagatagtg gaaaaggaag gtggctccta 60

caaatgccat cattgcgata aaggaaaggc catcgttgaa gatgcctctg ccgacagtgg 120

tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg ttccaaccac 180

gtcttcaaag caagtggatt gatgtgatat ctccactgac gtaagggatg acgcacaatc 240

ccactatcct tcgcaagacc cttcctctat ataaggaagt tcatttcatt tggagaggac 300

acgctgacaa gctgactcta gcagatctac cgtcttcggt acgcgctcac tccgccctct 360

gcctttgtta ctgccacgtt tctctgaatg ctctcttgtg tggtgattgc tgagagtggt 420

ttagctggat ctagaattac actctgaaat cgtgttctgc ctgtgctgat tacttgccgt 480

cctttgtagc agcaaaatat agggacatgg tagtacgaaa cgaagataga acctacacag 540

caatacgaga aatgtgtaat ttggtgctta gcggtattta tttaagcaca tgttggtgtt 600

atagggcact tggattcaga agtttgctgt taatttaggc acaggcttca tactacatgg 660

gtcaatagta tagggattca tattataggc gatactataa taatttgttc gtctgcagag 720

cttattattt gccaaaatta gatattccta ttctgttttt gtttgtgtgc tgttaaattg 780

ttaacgcctg aaggaataaa tataaatgac gaaattttga tgtttatctc tgctccttta 840

ttgtgaccat aagtcaagat cagatgcact tgttttaaat attgttgtct gaagaaataa 900

gtactgacag tattttgatg cattgatctg cttgtttgtt gtaacaaaat ttaaaaataa 960

agagtttcct ttttgttgct ctccttacct cctgatggta tctagtatct accaactgac 1020

actatattgc ttctctttac atacgtatct tgctcgatgc cttctcccta gtgttgacca 1080

gtgttactca catagtcttt gctcatttca ttgtaatgca gataccaagc ggcc 1134

<210>6

<211>253

<212>DNA

<213>nos nt

<400>6

gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60

atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120

atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180

gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240

atgttactag atc 253

<210>7

<211>552

<212>DNA

<213>cPAT NT

<400>7

atgtctccgg agaggagacc agttgagatt aggccagcta cagcagctga tatggccgcg 60

gtttgtgata tcgttaacca ttacattgag acgtctacag tgaactttag gacagagcca 120

caaacaccac aagagtggat tgatgatcta gagaggttgc aagatagata cccttggttg 180

gttgctgagg ttgagggtgt tgtggctggt attgcttacg ctgggccctg gaaggctagg 240

aacgcttacg attggacagt tgagagtact gtttacgtgt cacataggca tcaaaggttg 300

ggcctaggat ccacattgta cacacatttg cttaagtcta tggaggcgca aggttttaag 360

tctgtggttg ctgttatagg ccttccaaac gatccatctg ttaggttgca tgaggctttg 420

ggatacacag cccggggtac attgcgcgca gctggataca agcatggtgg atggcatgat 480

gttggttttt ggcaaaggga ttttgagttg ccagctcctc caaggccagt taggccagtt 540

acccagatct ga 552

<210>8

<211>1993

<212>DNA

<213>pUbi nt

<400>8

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660

cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720

acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780

ggcaggcggc ctcctcctcc tctcacggca ccggcagcta cgggggattc ctttcccacc 840

gctccttcgc tttcccttcc tcgcccgccg taataaatag acaccccctc cacaccctct 900

ttccccaacc tcgtgttgtt cggagcgcac acacacacaa ccagatctcc cccaaatcca 960

cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc cccccccccc cctctctacc 1020

ttctctagat cggcgttccg gtgcatggtt agggcccggt agttctactt ctgttcatgt 1080

ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag cgttcgtaca cggatgcgac 1140

ctgtacgtca gacacgttct gattgctaac ttgccagtgt ttctctttgg ggaatcctgg 1200

gatggctcta gccgttccgc agacgggatc gatttcatga ttttttttgt ttcgttgcat 1260

agggtttggt ttgccctttt cctttatttc aatatatgcc gtgcacttgt ttgtcgggtc 1320

atcttttcat gctttttttt gtcttggttg tgatgatgtg gtctggttgg gcggtcgttc 1380

tagatcggag tagatttctg tttcaaacta cctggtggat ttattaattt tggatctgta 1440

tgtgtgtgcc atacatattc atagttacga attgaagatg atggatggaa atatcgatct 1500

aggataggta tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt 1560

cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta gatcggagta 1620

gaatactgtt tcaaactacc tggtgtattt attaattttg gaactgtatg tgtgtgtcat 1680

acatcttcat agttacgagt ttaagatgga tggaaatatc gatctaggat aggtatacat 1740

gttgatgtgg gttttactga tgcatataca tgatggcata tgcagcatct attcatatgc 1800

tctaaccttg agtacctatc tattataata aacaagtatg ttttataatt attttgatct 1860

tgatatactt ggatgatggc atatgcagca gctatatgtg gattttttta gccctgcctt 1920

catacgctat ttatttgctt ggtactgttt cttttgtcga tgctcaccct gttgtttggt 1980

gttacttctg cag 1993

<210>9

<211>195

<212>DNA

<213>35s nt

<400>9

ctgaaatcac cagtctctct ctacaaatct atctctctct ataataatgt gtgagtagtt 60

cccagataag ggaattaggg ttcttatagg gtttcgctca tgtgttgagc atataagaaa 120

cccttagtat gtatttgtat ttgtaaaata cttctatcaa taaaatttct aattcctaaa 180

accaaaatcc agtgg 195

<210>10

<211>20

<212>DNA

<213>Cry1Fa-393F

<400>10

cgtcatccca acttacaagg 20

<210>11

<211>20

<212>DNA

<213>Cry1Fa-930R

<400>11

catgattcag cacatgggag 20

<210>12

<211>20

<212>DNA

<213>CF5

<400>12

cattcgcgta caccattgtc 20

<210>13

<211>20

<212>DNA

<213>cr5

<400>13

accagcaaag atccgttcac 20

<210>14

<211>23

<212>DNA

<213>18srRNA-F

<400>14

ccatccctcc gtagttagct tct 23

<210>15

<211>22

<212>DNA

<213>18srRNA-R

<400>15

cctgtcggcc aaggctatat ac 22

Claims

1. The protein for resisting the insect of the plant is characterized in that the amino acid sequence of the protein is SEQ ID NO. 3.

2. A pest-resistant gene comprising a gene sequence encoding the protein of claim 1.

3. The insect-resistant gene of claim 2 having the nucleotide sequence of SEQ ID NO 1.

4. An expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising the insect-resistant gene of claim 2 or 3.

5. An expression vector comprising the insect-resistant gene of claim 2.

6. The expression vector of claim 5, comprising the following gene structures in sequence:

from the maize ubiquitin gene promoter (Ubi); the insect-resistant gene of claim 2 or 3; a nopaline synthase terminator (Nos); encoding a phosphinothricin acetyltransferase gene (PAT); terminator from cauliflower mosaic virus (CaMV) (35 s).

7. Use of a protein according to claim 1 for plant pest resistance, or a pest resistance gene according to claim 2 or 3, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line comprising a pest resistance gene according to claim 2 or 3, in plant pest resistance, wherein the use is selected from one or both of: a) preparing a medicament having an effect of resisting cutworm and/or spodoptera frugiperda; b) breeding transgenic plants having or having increased resistance to cutworm and/or spodoptera frugiperda.

8. A method of growing plants having or having increased resistance to cutworm and/or spodoptera frugiperda, said method comprising the steps of: introducing the insect-resistant gene of claim 2 or 3 into a recipient plant to obtain a transgenic plant.

9. The method of claim 8, wherein the plant is selected from one or more of a monocot, a dicot, a gramineous plant; preferably corn.