CN111676233A

CN111676233A - Insect-resistant gene Cry1Ab-l and encoding protein and application thereof

Info

Publication number: CN111676233A
Application number: CN202010553538.0A
Authority: CN
Inventors: 刘允军; 刘艳; 王国英
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2020-09-18

Abstract

The invention provides an insect-resistant gene Cry1Ab-l and an encoding protein and application thereof. The sequences of the gene Cry1Ab-l and the protein coded by the gene Cry1Ab-l are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2. The indoor insecticidal test result shows that the insecticidal protein coded by the insect-resistant gene Cry1Ab-l has higher insecticidal activity than Cry1Ab, and can be used as a novel insecticidal protein. The resistance of transgenic Cry1Ab-l gene corn prepared by introducing the gene Cry1Ab-l into a plant body is obviously improved, and the transgenic Cry1Ab-l gene corn has high resistance to lepidoptera pests.

Description

Insect-resistant gene Cry1Ab-l and encoding protein and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to an insect-resistant gene Cry1Ab-l and an encoding protein and application thereof.

Background

Insect pests are a major cause of severe crop losses, and the most common means of controlling agricultural pests are the use of chemical and biological insecticides. Most chemical insecticides have the characteristics of broad spectrum and high toxicity, often kill a plurality of beneficial insects together while killing target pests, seriously damage ecological balance and cause serious pollution to the environment; in addition, pesticide residues pose a serious threat to the health of human beings and livestock. The biological insecticide has the characteristics of easy degradation and high compatibility with the environment, but needs to be repeatedly applied in production, thereby greatly increasing the production cost. In order to overcome the defects of chemical pesticides and biological pesticides in agricultural production, scientists introduce genes capable of coding insecticidal proteins into plants to cultivate various transgenic plants.

At present, hundreds of Bt insecticidal protein genes are cloned, but the insecticidal protein genes can be really applied to production but yield to fingers, and the reasons are mainly that the toxicity is low, the expression quantity in transgenic crops is low, and pests are easy to generate resistance; in addition, the increasing shortage of insecticidal protein gene cloning resources also causes great difficulty in gene cloning. Therefore, it is necessary to create new Bt genes with insecticidal activity and to use these genes to transform maize to create new transgenic maize materials.

Disclosure of Invention

The invention aims to provide an insect-resistant gene Cry1Ab-l and a coding protein and application thereof.

To achieve the object of the present invention, in a first aspect, the present invention provides an insect-resistant gene Cry1Ab-l, which is a gene encoding the following protein (a) or (b):

(a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 2;

(b) 2, protein which is derived from (a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 2.

The gene Cry1Ab-l is optimized according to the codon preference of corn, and the nucleotide sequence of the optimized gene Cry1Ab-l is shown as SEQ ID NO. 1.

In a second aspect, the present invention provides biomaterials containing the gene Cry1Ab-l, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, phage vectors, viral vectors, engineered bacteria, transgenic cell lines, or non-regenerable plant parts.

In a third aspect, the invention provides any one of the following uses of a protein encoded by the gene Cry1 Ab-l:

1) for the control of lepidopteran pests (in particular lepidopteran larvae);

2) for the preparation of insecticides;

in the invention, the lepidoptera pests are preferably corn borers, cotton bollworms and spodoptera frugiperda.

In a fourth aspect, the invention provides an insecticide, the effective component of which is protein encoded by gene Cry1 Ab-l.

In a fifth aspect, the present invention provides any one of the following uses of the gene Cry1Ab-l, or of biological material containing the gene Cry1 Ab-l:

1) for plant breeding;

2) for the preparation of transgenic plants.

Among them, the breeding aim is to impart insect resistance to plants.

In a sixth aspect, the invention provides application of the gene Cry1Ab-l or biological materials containing the gene Cry1Ab-l in preparing transgenic plants with insect resistance.

In the present invention, the insects include, but are not limited to, lepidopteran insects.

In a seventh aspect, the present invention provides a method for obtaining corn with insect resistance comprising:

1) causing corn to comprise the gene Cry1 Ab-l; or the like, or, alternatively,

2) the corn is made to express the protein coded by the gene Cry1 Ab-l.

Such methods include, but are not limited to, transgenics, crosses, backcrosses, selfs, or asexual propagation.

In an eighth aspect, the present invention provides a method for identifying a plant, wherein said plant is a plant comprising gene Cry1Ab-l, a plant expressing a protein encoded by gene Cry1Ab-l or a plant obtained according to the above method; the method comprises the following steps:

1) determining whether said plant comprises the gene Cry1 Ab-l; or the like, or, alternatively,

2) determining whether said plant expresses a protein encoded by the gene Cry1 Ab-l.

Compared with the prior art, the invention has at least the following advantages:

the invention selects a Cry1Ab protein truncated sequence, utilizes a corn preference codon to transform, creates a novel insect-resistant gene Cry1Ab-1, and greatly improves the expression quantity of Cry1Ab-l in transgenic corn and the insect resistance of transgenic Cry1Ab-1 gene corn.

The insecticidal protein coded by the (II) insect-resistant gene Cry1Ab-l has higher insecticidal activity than Cry1Ab, and can be used as a novel insecticidal protein.

And thirdly, the insect resistance of the corn transformed with Cry1Ab-l gene is obviously improved, and the corn has high resistance to lepidoptera pests.

Drawings

FIG. 1 is a plasmid map of a plant expression vector pCAMBIA3301 (modified plasmid pCAMBIA3301) in a preferred embodiment of the present invention.

FIG. 2 is a diagram of p3301Ubi-Cry1Ab plant expression vector of insecticidal protein gene Cry1Ab in the preferred embodiment of the present invention.

FIG. 3 is a diagram of p3301Ubi-Cry1Ab-l plant expression vector of insecticidal protein gene Cry1Ab-l in a preferred embodiment of the invention.

FIG. 4 shows the results of the identification of the transgenic corn indoor borers.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 engineering Synthesis of Cry1Ab-l Gene

The invention provides an insecticidal protein gene which has toxicity to corn borer and cotton bollworm and insecticidal activity to spodoptera frugiperda and a coding protein thereof.

1155 amino acid sequences of Cry1Ab protein (GenBank No. X04698) from Bacillus thuringiensis (Bt) are analyzed, and the first 658 amino acid residues are selected as novel insecticidal protein and named Cry1 Ab-l. The gene for coding Cry1Ab-l is optimized according to the codon preference of corn, the G + C content is increased to 63.7% from the original 38.8%, and the expression level of the Cry1Ab-l gene in the transgenic corn is further increased. The designed Cry1Ab-l gene was synthesized by Biotechnology engineering (Shanghai) Inc. The sequences of the gene Cry1Ab-l and the protein coded by the gene Cry1Ab-l are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.

Example 2 prokaryotic expression and Induction and purification of Cry1Ab and Cry1Ab-l proteins

Cry1Ab (SEQ ID NO:3) and Cry1Ab-l are synthesized by Shanghai and constructed on pET30a vector to obtain plasmids pET30-Cry1Ab and pET30-Cry1Ab-l with chloramphenicol resistance. The plasmid was transferred into a Transetta strain (DE3) purchased from Okinawa corporation, and a single clone was picked up and verified for positive plaque by PCR amplification. The positive plaque is inoculated into 10mL LB liquid culture medium (containing appropriate antibiotic), and is cultured with shaking overnight at 37 ℃ to obtain seed liquid.

The next day, according to 1: 200, inoculating the seed solution into 200mL LB liquid culture medium (containing proper antibiotics), culturing at 37 ℃ and 200rpm with shaking until OD600 is 0.4-0.6, adding IPTG to a final concentration of 0.5mM, culturing at 16 ℃ with shaking at 160rpm for about 20h with shaking, and inducing the expression of the target protein.

Collecting 200mL of induced thallus, centrifuging at 4 ℃ and 5000rpm for 5min, removing supernatant, adding 10mL of resuspension (25mM Tris-HCl, 150mM NaCl and 15mM imidazole), adding 10mg/mL of lysozyme to a final concentration of 100 mu g/mL, oscillating, placing on ice for 10min, carrying out ultrasonic crushing, carrying out ultrasonic treatment for 4s, 300w and 15-20min every 3s, fully crushing the thallus, and carrying out ice crushing operation during the crushing process.

Centrifuging at 12000rpm at 4 deg.C for 15min, collecting supernatant, and filtering with 0.45 μm filter membrane. 1mL of the mixed 50% Ni-NTA resin was added and the mixture was shaken on a shaker at room temperature for 90min to allow the target protein to be fully bound to the Ni-NTA resin. And (3) loading the Ni-NTA resin combined with the target protein into a column for purification. The nickel column was equilibrated with 70% ethanol in advance, and then equilibrated with 5 times the volume of the nickel column of the resuspension solution.

The well mixed protein supernatant and resin mixture was added to the column at low speed and the effluent collected and labeled as L15.

The effluent was collected with 8mL of solution I (25mM Tris-HCl, 150mM NaCl, 30mM imidazole), labeled L30.

The flow-through was collected with 4mL of solution II (25mM Tris-HCl, 150mM NaCl, 100mM imidazole), labeled L100.

The eluate was collected and labeled L250 by washing with 10mL of solution III (25mM Tris-HCl, 150mM NaCl, 250mM imidazole).

The purification process is operated in an environment of 4 ℃ and cross contamination is strictly avoided.

The purified proteins were transferred into dialysis bags, respectively, and dialyzed with a dialysate (25mM Tris-HCl, 150mM NaCl) at 4 ℃ for 24 hours with rotor agitation, with the dialysate being changed every 8 hours. After dialysis treatment, purified L250 protein was quantified using a Coomassie Brilliant blue protein quantification kit.

Example 3 indoor insecticidal test of Cry1Ab and Cry1Ab-l proteins

Indoor tests of corn borer, cotton bollworm and spodoptera frugiperda are carried out by adopting an artificial feed mixing method under the conditions of indoor temperature of 28 +/-1 ℃, photoperiod (L: D) of 16h:8h and relative humidity of 70-80%, purified protein is added into the artificial feed in an amount of 25 mu g/g to prepare a feed, the feed is uniformly distributed into 48-hole cell culture plates, and the feed without protein is used as negative control. And respectively inoculating one end of a first-hatched larva of corn borer, cotton bollworm or spodoptera frugiperda to each hole of different culture plates, inoculating 144 insects in total, and counting the insect mortality after 7 days. The result shows that the insecticidal effect of the insecticidal protein Cry1Ab-l on corn borer, cotton bollworm and spodoptera frugiperda is obviously improved compared with Cry1Ab (Table 1).

TABLE 1 insecticidal efficiency of prokaryotic expression of purified proteins

Protein species	Corn borer	Bollworm	Spodoptera frugiperda
				Cry1Ab	80％	75％	40％
Cry1Ab-l	100％	100％	70％
				Negative control	0％	0％	0％

Example 4 construction of transgenic maize

1. Construction of plant transformation vectors p3301Ubi-Cry1Ab and p3301Ubi-Cry1Ab-l

Plasmid pCAMBIA3301 was digested with HindIII and BamHI, and the 35S promoter was replaced with the maize Ubiquitin promoter to construct modified plasmid pCAMBIA3301 (FIG. 1). The plasmid pCAMBIA3301, pET30-Cry1Ab, pET30-Cry1Ab-l which is modified by BamHI and SacI double enzyme digestion is used for enzyme digestion for 1h in a water bath kettle at 37 ℃, and the enzyme digestion system is as follows:

carrying out agarose gel electrophoresis on the enzyme digestion product, respectively cutting a Cry1Ab fragment (SEQID NO:3) with the size of about 3.5kb, a Cry1Ab-l fragment with the size of 2kb and a modified pCAMBIA3301 vector sequence with the size of about 11kb by contrasting with a Marker, and recovering the nucleic acid fragment by using a gel recovery kit of Tiangen Biotechnology (Beijing) Limited company; then, the nucleic acid fragment recovered by cutting the gel is ligated with the vector fragment recovered by purification according to the instructions for use of T4 ligase. After the ligation reaction was completed, 5. mu.L of the ligation product was used to transform E.coli Trans 5. alpha. Selecting clone bacterial plaque in 10mL LB liquid culture medium containing corresponding antibiotic, shaking and culturing overnight at 37 deg.C, extracting plasmid and quantifying the next day. The plasmid was digested with BamHI and SacI, and the correctness of the construction of the vectors p3301Ubi-Cry1Ab (FIG. 2) and p3301Ubi-Cry1Ab-l (FIG. 3) was verified by agarose electrophoresis.

2. Transgenic corn plant obtained by agrobacterium transformation method

The vectors p3301Ubi-Cry1Ab and p3301Ubi-Cry1Ab-l were transformed into Agrobacterium EHA105 by freeze-thaw method for PCR identification. Taking a freshly peeled maize heald 31 young embryo with the diameter of about 1mm as a material, placing the young embryo in an infection culture medium for 1h, cleaning the young embryo once by using the infection culture medium, then soaking the young embryo in agrobacterium liquid of the infection culture medium added with 100 mu M acetosyringone, and placing 5 minutes. Taking out, drying with sterilized filter paper, placing on co-culture medium, co-culturing at 26 deg.C in dark for 3 days, and setting control. After the young embryos are washed and sterilized, the young embryos are placed on a screening culture medium containing 1.5mg/L of Bialaphos, screening culture is started for two weeks, then screening culture is carried out on the screening culture medium containing 3mg/L of Bialaphos, subculture is carried out every three weeks, screening culture is carried out for two months, and some calluses are good in growth state and are resistant calluses. The selected resistant callus is transferred to an embryoid induction culture medium, and embryoid can appear after 3 weeks. Then transferring the mixture to a differentiation medium for differentiation, wherein the culture condition is 28 ℃, the light intensity of 3000Lux every day, and the regenerated plantlets can appear quickly after 16 hours of illumination. When the regenerated plantlets grow to 3 leaves, the seedlings can be transplanted into cans and cultured indoors. After the young seedlings grow new leaves and roots, the young seedlings are taken out from a can bottle, the culture medium is flushed by tap water, and the young seedlings are transplanted into a small flowerpot mixed with nutrient soil and vermiculite (1:3, volume ratio). When the corn grows 2-3 new leaves, the corn can be transplanted into a field or a flowerpot for selfing to obtain T1 generation seeds.

The infection medium, co-culture medium, regeneration medium and rooting medium used in this example were as shown in ZL 201710090814.2.

Example 5 detection of expression levels of Cry1Ab and Cry1Ab-l in transgenic maize

Cry1Ab protein and Cry1Ab-l protein contents are detected by using a Cry1Ab/Cry1Ac protein ELISA detection kit (QualipateTM kit for Cry1Ab/Cry1Ac) produced by American ENVIROLOGIX company. Approximately 0.1g of transgenic maize leaves were weighed and ground to a powder in liquid nitrogen. Adding 1ml protein extract (from the ELISA detection kit), shaking and mixing for 30min to dissolve protein in the extract, centrifuging, and collecting supernatant for analysis. Adding 50ul of enzyme-linked reaction solution into each plate hole of the enzyme-labeled plate, then respectively adding Cry1Ab standard protein and transgenic corn protein extract with different concentrations into different holes, incubating for 1-2 hours at room temperature, washing for 3 times by using washing buffer solution, adding 100ul of reaction substrate, incubating for 10-30 minutes at room temperature, adding 100ul of termination buffer solution, measuring the light absorption value at 450nm by using an enzyme-labeled instrument, and calculating the contents of Cry1Ab protein and Cry1Ab-l protein in the transgenic corn. The results show that the Cry1Ab protein content in transgenic Cry1Ab corn is significantly higher than that of transgenic Cry1Ab-l (Table 2).

TABLE 2 protein content of interest in transgenic maize

Corn material	Content of the protein of interest
		Cry1Ab transgenic corn	Fresh weight of 3ug/g
Cry1Ab-l transgenic corn	Fresh weight of 7ug/g
		Non-transgenic corn heald 31	Fresh weight of 0ug/g

Example 6 identification of insect resistance in transgenic maize

When the transgenic corn planted in the greenhouse field is long and reaches the silking period, the sampled corn silks are placed into culture dishes, and 10 larvae of the primarily hatched corn borers are connected to each dish. The test is carried out in a culture chamber with relative humidity of 70% -80%, temperature of 26-28 deg.C and illumination period of 16h:8h (L: D), insect mortality is counted every 24h, new tissue from the same source is added or replaced according to tissue consumption, and the test is repeated three times under the same conditions. The results show that the non-transgenic corn heald 31 filament high-sensitivity corn borer, the transgenic Cry1Ab gene corn borer and the transgenic Cry1Ab-l gene corn have obviously improved insect resistance and high resistance (figure 4).

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> institute of crop science of Chinese academy of agricultural sciences

<120> insect-resistant gene Cry1Ab-l and encoding protein and application thereof

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tccaactcct ccgtgtccat catcagggcc ccgatgttct cctggataca caggtccgcc 1380

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

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

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

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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

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

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

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

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

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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

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

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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

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

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

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

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Asn Glu Leu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val

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

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cgaggctcgg ctcagggcat agaaggaagt attaggagtc cacatttgat ggatatactt 900

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ataatggctt ctcctgtagg gttttcgggg ccagaattca cttttccgct atatggaact 1020

atgggaaatg cagctccaca acaacgtatt gttgctcaac taggtcaggg cgtgtataga 1080

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ccacctaggc aaggatttag tcatcgatta agccatgttt caatgtttcg ttcaggcttt 1320

agtaatagta gtgtaagtat aataagagct cctatgttct cttggataca tcgtagtgct 1380

gaatttaata atataattcc ttcatcacaa attacacaaa tacctttaac aaaatctact 1440

aatcttggct ctggaacttc tgtcgttaaa ggaccaggat ttacaggagg agatattctt 1500

cgaagaactt cacctggcca gatttcaacc ttaagagtaa atattactgc accattatca 1560

caaagatatc gggtaagaat tcgctacgct tctaccacaa atttacaatt ccatacatca 1620

attgacggaa gacctattaa tcaggggaat ttttcagcaa ctatgagtag tgggagtaat 1680

ttacagtccg gaagctttag gactgtaggt tttactactc cgtttaactt ttcaaatgga 1740

tcaagtgtat ttacgttaag tgctcatgtc ttcaattcag gcaatgaagt ttatatagat 1800

cgaattgaat ttgttccggc agaagtaacc tttgaggcag aatatgattt agaaagagca 1860

caaaaggcgg tgaatgagct gtttacttct tccaatcaaa tcgggttaaa aacagatgtg 1920

acggattatc atattgatca agtatccaat ttagttgagt gtttatctga tgaattttgt 1980

ctggatgaaa aaaaagaatt gtccgagaaa gtcaaacatg cgaagcgact tagtgatgag 2040

cggaatttac ttcaagatcc aaactttaga gggatcaata gacaactaga ccgtggctgg 2100

agaggaagta cggatattac catccaagga ggcgatgacg tattcaaaga gaattacgtt 2160

acgctattgg gtacctttga tgagtgctac ttaacgtatt tatatcaaaa aatagatgag 2220

tcgaaattaa aagcctatac ccgttaccaa ttaagagggt atatcgaaga tagtcaagac 2280

ttagaaatct atttaattcg ctacaatgcc aaacacgaaa cagtaaatgt gccaggtacg 2340

ggttccttat ggcgcctttc agccccaagt ccaatcggaa aatgtgccca tcattcccat 2400

catttctcct tggacattga tgttggatgt acagacttaa atgaggactt aggtgtatgg 2460

gtgatattca agattaagac gcaagatggc catgcaagac taggaaatct agaatttctc 2520

gaagagaaac cattagtagg agaagcacta gctcgtgtga aaagagcgga gaaaaaatgg 2580

agagacaaac gtgaaaaatt ggaatgggaa acaaatattg tttataaaga ggcaaaagaa 2640

tctgtagatg ctttatttgt aaactctcaa tatgatagat tacaagcgga taccaacatc 2700

gcgatgattc atgcggcaga taaacgcgtt catagcattc gagaagctta tctgcctgag 2760

ctgtctgtga ttccgggtgt caatgcggct atttttgaag aattagaagg gcgtattttc 2820

actgcattct ccctatatga tgcgagaaat gtcattaaaa atggtgattt taataatggc 2880

ttatcctgct ggaacgtgaa agggcatgta gatgtagaag aacaaaacaa ccaccgttcg 2940

gtccttgttg ttccggaatg ggaagcagaa gtgtcacaag aagttcgtgt ctgtccgggt 3000

cgtggctata tccttcgtgt cacagcgtac aaggagggat atggagaagg ttgcgtaacc 3060

attcatgaga tcgagaacaa tacagacgaa ctgaagttta gcaactgtgt agaagaggaa3120

gtatatccaa acaacacggt aacgtgtaat gattatactg cgactcaaga agaatatgag 3180

ggtacgtaca cttctcgtaa tcgaggatat gacggagcct atgaaagcaa ttcttctgta 3240

ccagctgatt atgcatcagc ctatgaagaa aaagcatata cagatggacg aagagacaat 3300

ccttgtgaat ctaacagagg atatggggat tacacaccac taccagctgg ctatgtgaca 3360

aaagaattag agtacttccc agaaaccgat aaggtatgga ttgagatcgg agaaacggaa 3420

ggaacattca tcgtggacag cgtggaatta cttcttatgg aggaataa 3468

Claims

1. An insect-resistant gene Cry1Ab-l, characterized in that it is a gene encoding the following protein (a) or (b):

(a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 2;

2. The gene of claim 1, wherein the nucleotide sequence is represented by SEQ ID NO 1.

3. Biological material comprising the gene of claim 1 or 2, said biological material being a recombinant DNA, an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector, an engineered bacterium or a non-regenerable plant part.

4. Use of a protein encoded by the gene of claim 1 or 2, wherein the protein is selected from the group consisting of:

1) for the control of lepidopteran pests;

2) for the preparation of insecticides;

preferably, the lepidopteran pests are corn borers, cotton bollworms and spodoptera frugiperda.

5. A pesticidal agent, characterized in that the effective ingredient is a protein encoded by the gene of claim 1 or 2.

6. Use of the gene of claim 1 or 2, or the biomaterial of claim 3 for any one of the following applications:

1) for plant breeding;

2) for the preparation of transgenic plants.

7. Use of the gene of claim 1 or 2, or the biomaterial of claim 3, for the preparation of a transgenic plant with insect resistance; wherein the insect comprises a lepidopteran pest;

8. A method for obtaining corn with insect resistance,

1) causing maize to comprise the gene of claim 1 or 2; or the like, or, alternatively,

2) allowing maize to express a protein encoded by the gene of claim 1 or 2;

wherein the insect comprises a lepidopteran pest; preferably, the lepidopteran pests are corn borers, cotton bollworms and spodoptera frugiperda.

9. The method of claim 8, comprising transgenesis, crossing, backcrossing, selfing or asexual propagation.

10. A method for identifying a plant, wherein the plant is a plant comprising the gene of claim 1 or 2, a plant expressing a protein encoded by the gene of claim 1 or 2, or a plant obtained by the method of claim 8 or 9, comprising:

1) determining whether the plant comprises the gene of claim 1 or 2; or the like, or, alternatively,

2) determining whether said plant expresses a protein encoded by the gene of claim 1 or 2.