CN107474120B

CN107474120B - Artificially synthesized Bt insecticidal gene mcry1F for transgenic insect-resistant plants

Info

Publication number: CN107474120B
Application number: CN201710701955.3A
Authority: CN
Inventors: 赖锦盛; 赵海铭; 宋伟彬
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2017-08-16
Filing date: 2017-08-16
Publication date: 2020-08-18
Anticipated expiration: 2037-08-16
Also published as: CN107474120A

Abstract

The invention discloses a Bt insecticidal gene mcry1F artificially synthesized for transgenic insect-resistant plants. The Bt insecticidal gene mCry1F for the transgenic insect-resistant plants provided by the invention encodes mCry1F protein. The mCry1F protein is b1) or b2) or b 3): b1) the amino acid sequence is protein shown as a sequence 2 in a sequence table; b2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table; b3) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and is related to plant insect resistance. Experiments show that the mcry1F gene is easier to obtain a transformant with high expression quantity than the cry1F gene, and compared with the cry1F protein, the mcry1F protein has obviously improved insecticidal activity on corn borers, armyworms, cotton bollworms and dichocrocis punctiferalis, and has important popularization value.

Description

Artificially synthesized Bt insecticidal gene mcry1F for transgenic insect-resistant plants

Technical Field

The invention belongs to the technical field of biological control, and particularly relates to a Bt mcry1F gene for plant expression.

Background

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). Adang, M.J. from Bacillus thuringiensis in 1985

Cry1Ac gene is cloned in Bacillus thuringiensis, Wabiko, H. et al, 1986, clone 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.

The success of introducing kanamycin resistance gene into tobacco cells was announced by the university of washington in 1983, and the success of transferring soybean gene into sunflower was announced by the university of wisconsin in 4 months in the same year, which together marked the birth of plant transgenic technology. In 1986, the first transgenic plants (insect-resistant, herbicide-resistant) were approved for field trials. In 1989, mammalian antibody heavy and light chain genes were successfully expressed in tobacco and correctly assembled into functional antibodies. Gordon-Kamm in 1990 reported for the first time that fertile transformants were obtained by transforming maize suspension cell lines with a gene gun. Subsequently, the transgenic technology of corn began to develop rapidly, and a large number of transgenic plants were successively developed. Koziel (1993) and the like cultivate insect-resistant transgenic corn, and transgenic plants can horizontally express Cry1Ab insect-resistant genes, so that the insect resistance of the corn is improved. Zhang Xiujun (1999) et al used the particle gun method to introduce two genes containing high lysine protein into embryogenic callus of maize. Liu Da Wen (2000) and the like transform Zm13-Barnase gene into maize embryonic callus, and positive plants are obtained by screening herbicide. Ishida (1996) and the like establish the young embryo of the agrobacterium tumefaciens transformed maize inbred line by utilizing a super binary vector for the first time, and Frame (2002) and the like successfully realize the transformation of the young embryo by the agrobacterium tumefaciens by utilizing a common binary vector. Zhang Yan Zhen (2002) has systematically studied the introduction of Bt insecticidal genes into superior maize inbred lines by Agrobacterium tumefaciens mediated method. Huang and Wei (2005) successfully transformed conventional maize inbred lines with Agrobacterium tumefaciens. Conventional maize inbred lines were successfully transformed with agrobacterium respectively, Frame (2006) et al and Lee (2007).

In the aspect of transgenic technology research, a relatively mature corn transgenic technology system is established in China, corn genetic transformation work is earlier developed at China and China agricultural university, 1994, insect-resistant gene Bt is transferred into corn by an ovary injection method for the first time at home and abroad to obtain transgenic plants, and insect-resistant corn represented by the independent intellectual property gene Bt has shown good development prospect. Meanwhile, the method has stronger innovation capability in the aspects of core technology innovation such as no selective marker, selective marker gene deletion, target gene product timed degradation, plant tissue specific dominant expression and the like. Many insect-resistant genes are isolated at present, and the main insect-resistant genes are as follows: the insect-resistant genes derived from bacteria are mainly Bt toxalbumin genes (Cry1Ab, Cry1Ac, Cry2A, Cry3C and the like); secondly, the protease inhibitor gene from plants is characterized by wide insect-resistant spectrum and easy acceptance by the public from plants; ③ the nutritional insecticidal protein (Vip1, Vip2, Vip3, etc.) from bacteria, has broad-spectrum lepidoptera resistance, and especially has strong effects on black cutworm, armyworm and beet armyworm. Due to the influence of the insect resistance capability of the gene and the tolerance of insects, the transgenic insect-resistant product is mainly obtained by obtaining more effective insect-resistant genes, modifying the existing genes, double-resistant plant bodies and the like.

The United states has become the largest country for planting transgenic corn in the world, and the promotion area of the transgenic corn accounts for 25% of the total area of the American corn in 2000, and the promotion area of the transgenic corn accounts for 92% of the total area of the American corn in 2015. The data show that the use of Bt corn obviously reduces the use of pesticide and insecticide, and greatly reduces the pollution to the environment. Not only reduces the agricultural production cost, but also saves the labor force. The excellent insect-resistant character of the corn brings huge economic and environmental benefits to society, but the breeding of transgenic corn varieties in China has a larger gap than that in the United states, and thus, the 2008 nation starts the important special item of science and technology for breeding new varieties of transgenic organisms to improve the current situation. Although some reports are made in the aspect of breeding transgenic insect-resistant corn varieties in China, the transgenic insect-resistant corn varieties cannot be finally commercialized, and the results are mainly attributed to the insect-resistant effect of Bt genes and the stability of transformed plants.

Disclosure of Invention

The invention aims to solve the technical problem of how to improve the insect resistance of plants.

In order to solve the technical problems, the invention firstly provides the protein mCry 1F.

The protein of mCry1F provided by the invention can be b1) or b2) or b 3):

b1) the amino acid sequence is protein shown as a sequence 2 in a sequence table;

b2) a fusion protein obtained by connecting labels to the N end or/and the C end of the protein shown in the sequence 2 in the sequence table;

b3) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 2 in the sequence table and is related to plant insect resistance.

Wherein, the sequence 2 in the sequence table is composed of 600 amino acid residues.

In order to facilitate the purification of the protein in b1), the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 in the sequence table can be connected with a label shown in the table 1.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein of b3) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in b3) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein in b3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 1 in the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 above at the 5 'end and/or 3' end.

Nucleic acid molecules encoding the mCry1F protein are also within the scope of the invention.

The nucleic acid molecule encoding the mCry1F protein can be a DNA molecule shown as (a1) or (a2) or (a3) or (a4) as follows:

(a1) the coding region is a DNA molecule shown as a sequence 1 in a sequence table;

(a2) the nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table;

(a3) a DNA molecule having 75% or more 75% identity to the nucleotide sequence defined in (a1) or (a2) and encoding said mCry1F protein;

(a4) a DNA molecule which hybridizes with the nucleotide sequence defined in (a1) or (a2) under stringent conditions and encodes the mCry1F protein.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

Wherein, the sequence 1 in the sequence table is composed of 1803 nucleotides, and the nucleotide of the sequence 1 in the sequence table codes an amino acid sequence shown as a sequence 2 in the sequence table.

The nucleotide sequence encoding the mCry1F protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides that have been artificially modified to have 75% or greater identity to the nucleotide sequence of the mCry1F protein isolated according to the present invention, so long as they encode the mCry1F protein, are derived from and are identical to the nucleotide sequence of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of mCry1F protein consisting of the amino acid sequence shown in sequence 2 of the sequence listing of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

Expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines containing said nucleic acid molecules also belong to the scope of protection of the present invention.

The nucleotide sequence of the expression cassette is shown as a sequence 5 in a sequence table. In the sequence 5 in the sequence table, the 1 st to 583 rd positions from the 5' end are the nucleotide sequence of a maize Gly promoter, the 589 th to 2392 th positions are the nucleotide sequence of mcry1F gene, and the 2399 th to 2652 th positions are the nucleotide sequence of a nos terminator (for terminating transcription).

The recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule (namely a DNA molecule shown in a sequence 1 in a sequence table) for encoding the mCry1F protein into a starting plasmid.

The recombinant vector can be specifically a recombinant plasmid pCAMBIA3301+ mCry 1F. The recombinant plasmid pCAMBIA3301+ mCry1F is a double-stranded DNA molecule which is represented by 1 st to 2392 th sites from 5' end of a sequence 5 in a sequence table and replaces a small fragment between recognition sequences of restriction enzymes HindIII and BstEII of the vector pCAMBIA 3301. The recombinant plasmid pCAMBIA3301+ mCry1F expresses mCry1F protein shown in sequence 2 in the sequence table.

The recombinant microorganism can be obtained by introducing the recombinant vector into the starting microorganism.

The starting microorganism may be a yeast, bacterium, algae or fungus. The bacteria may be gram positive or gram negative bacteria. The gram-negative bacterium may be agrobacterium tumefaciens (agrobacterium tumefaciens). The Agrobacterium tumefaciens (Agrobacterium tumefaciens) may specifically be Agrobacterium tumefaciens EHA 105.

The recombinant microorganism may specifically be EHA105/pCAMBIA3301+ mCry 1F. EHA105/pCAMBIA3301+ mCry1F is a recombinant Agrobacterium tumefaciens obtained by transforming Agrobacterium tumefaciens EHA105 with recombinant plasmid pCAMBIA3301+ mCry 1F.

None of the transgenic plant cell lines includes propagation material. The transgenic plant is understood to comprise not only the first generation transgenic plant obtained by transforming a recipient plant with said mCry1F gene (i.e. the gene encoding the mCry1F protein), but also the progeny thereof. For transgenic plants, the gene can be propagated in the species, and can also be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

The application of the mCry1F protein, or the nucleic acid molecule, or an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line containing the nucleic acid molecule also belongs to the protection scope of the invention; the mCry1F protein, or the nucleic acid molecule, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule may be used in e1) or e2) or e 3): e1) improving the insect resistance of the plants; e2) preparing a product with an insect-resistant effect; e3) cultivating the transgenic plant with improved insect resistance.

In the above application, the product may be a medicament.

In order to solve the technical problems, the invention also provides a method for cultivating the transgenic plant.

The method for cultivating the transgenic plant provided by the invention can comprise the following steps: introducing a nucleic acid molecule encoding the mCry1F protein into a receptor plant to obtain a transgenic plant; the transgenic plant has increased insect resistance as compared to the recipient plant.

In the above method, the nucleic acid molecule encoding the mCry1F protein may be a DNA molecule represented by (a1) or (a2) or (a3) or (a4) as follows:

In the above method, the "introducing a nucleic acid molecule encoding the mCry1F protein into a recipient plant" may be performed by introducing a recombinant vector into a recipient plant; the recombinant vector may be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the mCry1F protein into an expression vector.

The recombinant vector can be specifically the recombinant plasmid pCAMBIA3301+ mCry 1F.

In order to solve the technical problems, the invention also provides a plant breeding method.

The plant breeding method provided by the invention can comprise the following steps: increasing the content and/or activity of the mCry1F protein in a plant, thereby increasing insect resistance.

In the plant breeding method, the effect of increasing the content and/or activity of the protein mCry1F in the plant can be achieved by methods well known in the art, such as multiple copies, change of promoters, regulatory factors, transgenes and the like.

Any of the plants described above may be any of c1) to c 5): c1) a monocot plant; c2) a dicotyledonous plant; c3) a gramineous plant; c4) corn; c5) maize variety X178.

Any of the above insect resistances may be against lepidopteran insects. The lepidopteran insect may be any one of d1) to d 5): d1) corn borer; d2) oriental armyworm; d3) cotton bollworm; d4) dichocrocis punctiferalis; d5) asiatic corn borer.

The Cry1F gene has strong toxicity to lepidoptera insects (such as corn borers, armyworms, cotton bollworms and the like), but the original gene cannot be efficiently expressed in plants, so that the requirement of plant protection cannot be met. The inventor of the invention carries out detailed analysis on the active region of the Cry1F protein, and carries out gene coding frame and codon modification on the Cry1F protein according to the coding characteristics of monocotyledons on the premise of ensuring that the insecticidal activity of the protein is further improved. The modified Cry1F protein is named mcry1F protein. The amino acid sequence of the mcry1F protein is shown as a sequence 2 in a sequence table. The nucleotide sequence of the gene (namely the modified Cry1F gene, hereinafter abbreviated as mcry1F gene) for encoding the mcry1F protein is shown as a sequence 1 in a sequence table. The inventor connects the modified mcry1F gene to an expression vector taking corn Gly as a promoter. The mcry1F gene is transformed into a maize inbred line with high transformation efficiency by an agrobacterium transformation method. Experiments show that the transformant with high expression quantity can be obtained more easily by using the codon-optimized mcry1F gene than before codon optimization, and compared with the cry1F protein, the activity of the mcry1F protein in killing the corn borers, the armyworms, the cotton bollworms and the dichocrocis punctiferalis is obviously improved, so that the method has important popularization value.

Drawings

FIG. 1 shows the results of the experiment in step five of example 3.

FIG. 2 shows the results of the sixth step of example 3.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Solute of N6E culture medium and its concentration are N6 salt of 4g/L, N6vitamin Stock of 5mL/L (200X), 2 mg/L2, 4-D, 0.1g/L inositol, 2.76g/L proline, 30g/L sucrose, 0.1g/L casein hydrolysate, 2.8g/L phytogel and 3.4mg/L silver nitrate; the solvent is distilled water; the pH was 5.8.

N6vitamin Stock (200 ×) containing glycine 0.4g/L and nicotinic acid 0.1g/L, VB₁0.2g/L and VB₆0.1g/L of an aqueous solution.

N6E solid plate: N6E medium at about 55 ℃ was poured into a petri dish and cooled to give a N6E solid plate.

And (3) dip-dyeing a culture medium: sucrose (68.4 g), N6 (50 mL in bulk (20X)), B5 (10 mL in trace (100X)), N6 iron salt (100X), RTV organic (200X) 5mL and 100. mu. mol Acetosyringone (AS) were dissolved in 1L of distilled water, and the pH was adjusted to 5.2.

N6A large number (20 ×) of₄)₂SO₄9.26g/L、KNO₃56.60g/L、KH₂PO₄8.00g/L、MgSO₄·7H₂O3.70 g/L and CaCl₂·2H₂O3.32 g/L aqueous solution.

Trace B5 (100 ×) containing MnSO₄·H₂O 0.7600g/L、ZnSO₄·7H₂O 0.2000g/L、H₃BO₃0.3000g/L、KI 0.0750g/L、Na₂MoO₄·2H₂O 0.0250g/L、CuSO₄·5H₂O0.0025 g/L and CoCl₂·6H₂O0.0025 g/L of water solution.

N6 iron salt (100 ×): 1.8300g/L of sodium ferric ethylenediamine tetracetate.

RTV organic (200 ×) prepared by mixing 0.0196g of choline chloride and VB₂0.0098g, 0.0200g of D-biotin, 0.0400g of nicotinic acid, 0.0097g of folic acid and VB₁0.0944g, 0.0200g of calcium D-pantothenate, VB₆0.0400g, 0.0098g p-aminobenzoic acid and 400 μ L VB with a concentration of 0.75mg/100mL₁₂The aqueous solution was dissolved in 1L of distilled water.

Co-culture medium: 4.33g of MS salt, 2mL of MS Vitamins (500X), 0.5mg of thiamine hydrochloride, 30.0g of sucrose, 1.38g of L-proline, 0.5mg of 2,4-D, 0.01mg of 6-BA, 3.5g of vegetable gel and 100. mu. mol of AS were dissolved in 1L of distilled water, and the pH was adjusted to 5.7.

MS Vitamins (500 ×) containing glycine 1g/L and nicotinic acid 0.25g/L, VB₁0.05g/L and VB₆0.25g/L of an aqueous solution.

Recovering culture medium prepared from MS salt 4.33g, MS Vitamins (500 ×)2mL, thiamine hydrochloride 0.5mg, sucrose 30.0g, L-proline 1.38g, 2, 4-D0.5 mg, 6-BA 0.01mg, plant gel 3.5g, Tim 100mg, bialaphos 3.0mg and AgNO₃3.4mg were dissolved in 1L of distilled water and the pH was adjusted to 5.7.

Primary selection of culture medium: MS solid culture medium containing 1.5mg/L bialaphos.

Secondary selection of culture medium: MS solid culture medium containing 3.0mg/L bialaphos.

Regeneration medium I: 4.33g of MS salt, 2mL of MS Vitamins (500X), 0.5mg of thiamine hydrochloride, 10.0g of sucrose, 20g of glucose, 0.7g of L-proline, 3.5g of vegetable gel, 0.2g of casein hydrolysate, 0.04g of glycine, 0.1g of inositol, and 3.0mg of bialaphos were dissolved in 1L of distilled water, and the pH was adjusted to 5.7.

And (3) regeneration medium II: MS salt 2.165g, sucrose 30.0g, plant gel 3.5g and bialaphos 3.0mg were dissolved in 1L of distilled water, and the pH was adjusted to 5.7.

The prokaryotic expression vector pEASY-E1 is a product of Beijing Quanji gold biotechnology, Inc.

Example 1 modification of Cry1F Gene and acquisition of mcry1F Gene

The amino acid sequence of the Cry1F protein is shown as a sequence 4 in a sequence table. The nucleotide sequence of the gene for coding the Cry1F protein (hereinafter, abbreviated as Cry1F gene) is shown as a sequence 3 in a sequence table.

The inventor of the invention carries out careful analysis on the active region of the Cry1F protein, and carries out gene coding frame and codon modification on the Cry1F protein according to the coding characteristics of monocotyledons on the premise of ensuring that the expression level is further improved. The modified Cry1F protein is named mcry1F protein. The amino acid sequence of the mcry1F protein is shown as a sequence 2 in a sequence table. The nucleotide sequence of the gene (namely the modified Cry1F gene, hereinafter abbreviated as mcry1F gene) for encoding the mcry1F protein is shown as a sequence 1 in a sequence table.

The sequence homology of the mcry1F gene and the Cry1F gene is only 66%, the homologous region is only 51%, the length of the coding frame is changed from 1174 amino acids to 600 amino acids, the content of G + C is also changed from 38.9% to 66.6%, and the codon usage rates before and after modification are detailed in Table 2.

TABLE 2

Example 2 bioassay of Mcry1F protein on Lepidoptera insects

In vitro expression and purification of mcry1F protein

The in vitro expression and purification steps of the mcry1F protein are as follows:

1. artificially synthesizing a double-stranded DNA molecule shown as a sequence 1 in a 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-mcry 1F.

The recombinant plasmid pEASY-mcry1F was sequenced. The sequencing result shows that the recombinant plasmid pEASY-mcry1F contains DNA molecules shown in a sequence 1 in a sequence table, and expresses mcry1F protein shown in a sequence 2 in the sequence table.

3. The recombinant plasmid pEASY-mcry1F is introduced into escherichia coli transetta to obtain a recombinant bacterium, and the recombinant bacterium is named as transetta-mcry 1F.

4. A single clone of transetta-mcry1F 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 culture broth into 50mL LB liquid medium (containing 50. mu.g/mL ampicillin) at a volume ratio of 1:100, and performing shaking culture at 37 deg.C and 200rpm to OD_600nmThe value was 0.6, IPTG was added to the cells so that the concentration was 1mM, the cells were cultured with shaking at 28 ℃ and 220rpm for 4 hours, and the cells were centrifuged at 4 ℃ and 10000rpm for 10 minutes to collect cell pellets.

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 by using a nickel column manufactured by GE (the specific steps of the purification were referred to the specifications of the nickel column), and then mcry1F protein was quantified by using a protein quantification kit manufactured by Saimer Feishel.

According to the method, the double-stranded DNA molecule shown in the sequence 1 in the artificially synthesized sequence table in the step 1 is replaced by the double-stranded DNA molecule shown in the sequence 3 in the artificially synthesized sequence table, and other steps are not changed, so that cry1F protein is obtained.

Bioassay of double, mcry1F protein on Lepidoptera pests

The activity of mcry1F protein and cry1F protein were compared according to the bioassay method reported by sting Xue (2008), and the insecticidal activity of the two proteins (mcry1F protein and cry1F protein) against four lepidopteran insects (asian corn borer, oriental armyworm, cotton bollworm and dichocrocis punctifera) was determined separately.

Some experimental results are shown in table 3. The result shows that the insecticidal activity of mcry1F protein to four lepidoptera insects is obviously better than that of cry1F protein, and the insecticidal activity of cry1F gene to Asian corn borer, cotton bollworm, armyworm and dichocrocis punctiferalis is obviously improved.

TABLE 3

Example 3 obtaining of transgenic maize with mcry1F Gene and identification of insect resistance

First, construction of recombinant vector

Recombinant plasmid pCAMBIA3301+ mcry1F was constructed and sequenced. According to the sequencing results, the recombinant plasmid pCAMBIA3301+ mcry1F was structurally described as follows: a small fragment between recognition sequences of restriction enzymes HindIII and BstEII of a vector pCAMBIA3301 is replaced by a double-stranded DNA molecule shown in the 1 st to 2392 nd positions from the 5' end of a sequence 5 in a sequence table. The recombinant plasmid pCAMBIA3301+ mcry1F expresses mcry1F protein shown in sequence 2 in the sequence table.

The recombinant plasmid pCAMBIA3301+ mcry1F contains an expression cassette, and the nucleotide sequence of the expression cassette is shown as a sequence 5 in a sequence table. In the sequence 5 in the sequence table, the 1 st to 583 rd positions from the 5' end are the nucleotide sequence of a maize Gly promoter, the 589 th to 2392 th positions are the nucleotide sequence of mcry1F gene, and the 2399 th to 2652 th positions are the nucleotide sequence of a nos terminator (for terminating transcription).

The nucleotide sequence of mcry1F gene in the recombinant plasmid pCAMBIA3301+ mcry1F is replaced by the nucleotide sequence of Cry1F gene, and other sequences are not changed, so that the recombinant plasmid pCAMBIA3301+ Cry1F is obtained. The recombinant plasmid pCAMBIA3301+ mcry1F expresses cry1F protein shown in sequence 4 in the sequence table.

II, obtaining recombinant agrobacterium

The recombinant plasmid pCAMBIA3301+ mcry1F was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which was designated EHA105/pCAMBIA3301+ mcry 1F.

The recombinant plasmid pCAMBIA3301 was introduced into Agrobacterium tumefaciens EHA105 to obtain a recombinant Agrobacterium, which was designated as EHA105/pCAMBIA 3301.

The recombinant plasmid pCAMBIA3301+ Cry1F was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which was designated EHA105/pCAMBIA3301+ Cry 1F.

Obtaining of Mcry1F transgenic corn

1. Obtaining and culturing of immature embryos

(a) Planting a corn variety X178 in a field, removing bracts of pollinated clusters after self-pollination is carried out for 9-11 days, putting the clusters into a beaker filled with disinfectant (obtained by adding a drop of Tween 20 into 700mL of 50% (v/v) bleach aqueous solution or sodium hypochlorite aqueous solution (effective chlorine is 5.25% (v/v)) for 20min, and then washing with sterile water for 3 times. In the soaking process, the clusters need to be rotated at intervals and the beaker needs to be tapped lightly to remove air bubbles on the surface of the seeds, so that the optimal disinfection effect is achieved.

(b) After the step (a) is finished, picking up the fruit cluster, inserting the knife tip of the embryo peeling knife between the embryo and the endosperm, slightly prying out the immature embryo, slightly supporting the immature embryo by using a small surgical knife tip to ensure that the immature embryo is not damaged, and tightly attaching the embryo axial surface of the immature embryo to an N6E solid flat plate placed with filter paper, wherein the density of the immature embryo is about 2cm multiplied by 2cm (30 embryos per dish).

(c) After the step (b) is finished, taking the N6E solid plate, sealing the plate by using a sealing film, and culturing the plate in the dark at the temperature of 28 ℃ for 2-3 d.

2. Obtaining of Agrobacterium Dip dye

(a) EHA105/pCAMBIA3301+ mcry1F was inoculated on YEP solid medium containing 33mg/L Kanamycin (Kanamycin, Kana) and 50mg/L streptomycin (strymycin, str), cultured at 19 ℃ for 3 days, and activated.

(b) Inoculating EHA105/pCAMBIA3301+ mcry1F obtained in step (a) into a staining medium, and culturing at 25 deg.C and 75rpm with shaking to obtain OD_550nm0.3-0.4 of agrobacteria staining solution.

3. Obtaining of Mcry1F transgenic corn

The conditions of light-dark alternate culture (i.e., light culture and dark culture alternate) are as follows: at 25 ℃. The light intensity in the light culture was 15000 Lx. The period of light-dark alternate culture is specifically as follows: 16h light culture/8 h dark culture.

(a) And (3) putting the immature embryos which are subjected to the step 1 into a centrifuge tube, washing the immature embryos for 2 times by using a dip dyeing culture medium (2 mL of the dip dyeing culture medium is used each time), adding an agrobacterium-mediated dip dyeing solution, slightly inverting the centrifuge tube for 20 times, standing the immature embryos in a dark box for 5min vertically (ensuring that the immature embryos are completely soaked in the agrobacterium-mediated dip dyeing solution).

(b) After completion of step (a), the young embryos are transferred to a co-cultivation medium (the embryonic axis of the young embryos is brought into contact with the surface of the co-cultivation medium while removing the excess agrobacterium on the surface of the co-cultivation medium), and then cultured in the dark at 20 ℃ for 3 d.

(c) After completion of step (b), the young embryos are transferred to recovery medium and then cultured in the dark at 28 ℃ for 7 d.

(d) After completion of step (c), the young embryos are transferred to a primary selection medium and then cultured alternately in the dark and light at 28 ℃ for two weeks.

(e) After completion of step (d), the young embryos are transferred to a secondary selection medium and then cultured alternately in the dark and light at 28 ℃ for two weeks to obtain resistant calli.

(f) After completion of step (e), the resistant calli were transferred to regeneration medium I and then cultured alternately in the dark and light at 28 ℃ for three weeks.

(g) And (f) after the step (f) is finished, transferring the resistant callus to a regeneration culture medium II, and then alternately culturing for three weeks in the dark and the light at the temperature of 28 ℃ to obtain a regeneration seedling. Transferring the regenerated seedlings to a greenhouse when the regenerated seedlings grow to 3-4 leaves, and normally culturing to obtain the mcry1F transgenic corn. The 5 mcry1F transgenic corns are named mcry1F-1 to mcry1F-5 in sequence.

Following the above procedure, EHA105/pCAMBIA3301+ mcry1F was replaced with EHA105/pCAMBIA3301, and the other steps were unchanged to give empty vector maize.

EHA105/pCAMBIA3301+ mcry1F was replaced with EHA105/pCAMBIA3301+ Cry1F according to the above method, and the other steps were not changed to obtain transgenic Cry1F maize. The 5 transgenic Cry1F corns are sequentially named as Cry1F-1 to Cry 1F-5.

Fourth, molecular identification

Genomic DNAs of leaves of mcry1F-1 to mcry1F-5 were extracted and used as templates, respectively, and primers 1 FF: 5'-ACAACCACTACAACCGCCTCATC-3' and primer 1 FR: 5'-TGTTGATGGTGGCGTAGGAGAAG-3' to obtain PCR product A. Genomic DNAs of leaves of Cry1F-1 to Cry1F-5 were extracted and used as templates, and primers Cry1 FF: 5'-TATCGTTTGGGCAGGGTTGGG-3' and primer cry1 FR: 5'-CGCCACCAGGATTGAAGACCC-3' to obtain PCR product B.

The genomic DNA of the leaf of mcry1F-1 was replaced with water according to the above method, and the other steps were the same, and used as a negative control.

The genomic DNA of the leaf of mcry1F-1 was replaced with the genomic DNA of the leaf of empty vector maize according to the above method, and the same procedure was used as control 1.

The genomic DNA of the leaf of mcry1F-1 was replaced with the genomic DNA of the leaf of maize variety X178 according to the above method, and the same procedure was used as control 2.

The genomic DNA of the leaf of mcry1F-1 was replaced with the recombinant plasmid pCAMBIA3301+ mcry1F in the same manner as above, and used as a positive control 1.

The genomic DNA of the leaf of mcry1F-1 was replaced with the recombinant plasmid pCAMBIA3301+ Cry1F according to the above method, and the other steps were the same, as a positive control 2.

The PCR amplification product was subjected to agarose gel electrophoresis. The results show that the 1134bp strip can be obtained by amplification by using the genomic DNA of the leaves of mcry1F-1 to mcry1F-5 or the recombinant plasmid pCAMBIA3301+ mcry1F as a template, the 510bp strip can be obtained by using the genomic DNA of the leaves of Cry1F-1 to Cry1F-5 or the recombinant plasmid pCAMBIA3301+ Cry1F as a template, and the 1134bp strip or the 510bp strip can not be obtained by amplification by using water, the genomic DNA of the leaves of empty vector corn or the genomic DNA of the leaves of corn variety X178 as a template.

Through molecular identification, mcry1F-1 to mcry1F-5 are all transgenic mcry1F corns, and Cry1F-1 to Cry1F-5 are all transgenic Cry1F corns.

Fifthly, detecting the content of Cry1F protein in mcry1F transgenic corn

The sample to be tested is a leaf of mcry1F-1, a leaf of mcry1F-2, a leaf of mcry1F-3, a leaf of mcry1F-4, a leaf of mcry1F-5, a leaf of Cry1F-1, a leaf of Cry1F-2, a leaf of Cry1F-3, a leaf of Cry1F-4, a leaf of Cry1F-5, a leaf of empty-vector-transferred corn or a leaf of corn variety X178.

The Bt Cry1F ELISA kit is a product of Agdia company in the United states.

The experiment was repeated three times and the mean value was taken. The method comprises the following specific steps:

(1) sample preparation and positive control: grinding a sample to be detected by liquid nitrogen, weighing 0.1g of the ground sample, adding 1mL of sample extraction buffer solution (carried by the kit), diluting positive control carried by the kit by using 2mL of sample extraction buffer solution, fully mixing uniformly, and then centrifuging at a low speed for 30s to prepare for sample adding.

(2) The antibody (kit) was diluted with an enzyme-linked buffer (kit) at a ratio of 100:1, and the diluted antibody was added to an microplate at a volume of 100. mu.l per well. Adding 10 mu L of each different sample into the corresponding sample well, simultaneously adding 0 mu L, 5 mu L, 10 mu L, 15 mu L, 20 mu L, 25 mu L, 50 mu L and 100 mu L of positive control into the corresponding sample well, supplementing each sample well to 200 mu L by using plate washing buffer (carried by the kit), and incubating for 1h or overnight at 4 ℃ in a humid environment at room temperature.

(3) Washing the plate: washing the plate with 1 XPBST (kit with itself) for 7 times, adding 300 mu L of plate washing buffer solution each time, pouring out after filling one plate, inverting the ELISA plate after washing, and fully removing the residual liquid in the ELISA plate.

(4) Color development: mu.L of TMB color buffer was added to each well and incubated for 20min at room temperature in a humid environment.

Analyzing the light absorption values of different samples by a Thermo MK3 enzyme-labeling instrument at a wavelength of 650nm, carrying out Cry1F protein quantification by using a positive control drawn standard curve, and then counting the proportion of Cry1F protein in the fresh weight of the sample to be detected, namely obtaining the content of Cry1F protein.

The absorbance of a portion of the sample at 650nm is shown in FIG. 1. The results show that Cry1F protein can be detected in the leaves of mcry1F-1 to mcry1F-5 and the leaves of Cry1F-1 to Cry1F-5, and Cry1F protein can not be detected in the leaves of empty carrier corn and the leaves of corn variety X178. Meanwhile, compared with Cry1F-1 to Cry1F-5, the content of Cry1F protein in the leaves of mcry1F-1 to mcry1F-5 is improved to different degrees.

Six, resistance identification of Mcry1F transgenic corn

The corn to be detected is mcry1F-1, mcry1F-2, mcry1F-3, mcry1F-4, mcry1F-5, Cry1F-1, Cry1F-2, Cry1F-3, Cry1F-4, Cry1F-5, empty vector transferred corn or corn variety X178.

The experiment is set to be at least 2 times of biological repetition, each corn to be detected is repeatedly set to 2 parallel experiments, and the specific steps are as follows:

1. collecting leaves of the corn to be detected and putting the leaves into a culture dish.

2. After the step 1 is finished, 3 larvae of the primarily hatched corn borers are inoculated into each culture dish, and then the culture dish is placed at the temperature of 28 ℃ for 16h in a photoperiod: culturing in a climatic incubator with relative humidity of 70-80% for 8h (L: D). After 144h, the leaves were observed for phenotype.

The results of the experiment are shown in FIG. 2. The result shows that compared with the CRY1F transgenic corn, the leaf insect resistance of the transgenic corn with the mcry1F gene is obviously improved.

<110> university of agriculture in China

<120> artificially synthesized Bt insecticidal gene mcry1F for transgenic insect-resistant plants

<160>5

<170>PatentInversion3.5

<210>1

<211>1803

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>1

atggagaaca acatccagaa ccagtgcgtc ccctacaact gcctcaacaa ccccgaggtc 60

gagatcctca acgaggagcg ctccaccggc cgcctccccc tcgacatctc cctctccctc 120

acccgcttcc tcctctccga gttcgtcccc ggcgtcggcg tcgccttcgg cctcttcgac 180

ctcatctggg gcttcatcac cccctccgac tggtccctct tcctcctcca gatcgagcag 240

ctcatcgagc agcgcatcga gaccctcgag cgcaaccgcg ccatcaccac cctccgcggc 300

ctcgccgact cctacgagat ttacatcgag gccctccgcg agtgggaggc caaccccaac 360

aacgcccagc tccgcgagga cgtccgcatc cgcttcgcca acaccgacga cgccctcatc 420

accgccatca acaacttcac cctcacctcc ttcgagatcc ccctcctctc cgtctacgtc 480

caggccgcca acctccacct ctccctcctc cgcgacgccg tctccttcgg ccagggctgg 540

ggcctcgaca tcgccaccgt caacaaccac tacaaccgcc tcatcaacct catccaccgc 600

tacaccaagc actgcctcga cacctacaac cagggcctcg agaacctccg cggcaccaac 660

acccgccagt gggcccgctt caaccagttc cgccgcgacc tcaccctcac cgtcctcgac 720

atcgtcgccc tcttccccaa ctacgacgtc cgcacctacc ccatccagac ctcctcccag 780

ctcacccgcg agatttacac ctcctccgtc atcgaggact cccccgtctc cgccaacatc 840

cccaacggct tcaaccgcgc cgagttcggc gtccgccccc cccacctcat ggacttcatg 900

aactccctct tcgtcaccgc cgagaccgtc cgctcccaga ccgtctgggg cggccacctc 960

gtctcctccc gcaacaccgc cggcaaccgc atcaacttcc cctcctacgg cgtcttcaac 1020

cccggcggcg ccatctggat cgccgacgag gacccccgcc ccttctaccg caccctctcc 1080

gaccccgtct tcgtccgcgg cggcttcggc aacccccact acgtcctcgg cctccgcggc 1140

gtcgccttcc agcagaccgg caccaaccac acccgcacct tccgcaactc cggcaccatc 1200

gactccctcg acgagatccc cccccaggac aactccggcg ccccctggaa cgactactcc 1260

cacgtcctca accacgtcac cttcgtccgc tggcccggcg agatttccgg ctccgactcc 1320

tggcgcgccc ccatgttctc ctggacccac cgctccgcca cccccaccaa caccatcgac 1380

cccgagcgca tcacccagat ccccctcgtc aaggcccaca ccctccagtc cggcaccacc 1440

gtcgtccgcg gccccggctt caccggcggc gacatcctcc gccgcacctc cggcggcccc 1500

ttcgcctaca ccatcgtcaa catcaacggc cagctccccc agcgctaccg cgcccgcatc 1560

cgctacgcct ccaccaccaa cctccgcatc tacgtcaccg tcgccggcga gcgcatcttc 1620

gccggccagt tcaacaagac tatggacacc ggcgaccccc tcaccttcca gtccttctcc 1680

tacgccacca tcaacaccgc cttcaccttc cccatgtccc agtcctcctt caccgtcggc 1740

gccgacacct tctcctccgg caacgaggtc tacatcgacc gcttcgagct gatccccgtc 1800

tga 1803

<210>2

<211>600

<212>PRT

<213> Artificial sequence

<220>

<223>

<400>2

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

595 600

<210>3

<211>3525

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>3

atggagaata atattcaaaa tcaatgcgta ccttacaatt gtttaaataa tcctgaagta 60

gaaatattaa atgaagaaag aagtactggc agattaccgt tagatatatc cttatcgctt 120

acacgtttcc ttttgagtga atttgttcca ggtgtgggag ttgcgtttgg attatttgat 180

ttaatatggg gttttataac tccttctgat tggagcttat ttcttttaca gattgaacaa 240

ttgattgagc aaagaataga aacattggaa aggaaccggg caattactac attacgaggg 300

ttagcagata gctatgaaat ttatattgaa gcactaagag agtgggaagc aaatcctaat 360

aatgcacaat taagggaaga tgtgcgtatt cgatttgcta atacagacga cgctttaata 420

acagcaataa ataattttac acttacaagt tttgaaatcc ctcttttatc ggtctatgtt 480

caagcggcga atttacattt atcactatta agagacgctg tatcgtttgg gcagggttgg 540

ggactggata tagctactgt taataatcat tataatagat taataaatct tattcataga 600

tatacgaaac attgtttgga cacatacaat caaggattag aaaacttaag aggtactaat 660

actcgacaat gggcaagatt caatcagttt aggagagatt taacacttac tgtattagat 720

atcgttgctc tttttccgaa ctacgatgtt agaacatatc caattcaaac gtcatcccaa 780

ttaacaaggg aaatttatac aagttcagta attgaggatt ctccagtttc tgctaatata 840

cctaatggtt ttaatagggc ggaatttgga gttagaccgc cccatcttat ggactttatg 900

aattctttgt ttgtaactgc agagactgtt agaagtcaaa ctgtgtgggg aggacactta 960

gttagttcac gaaatacggc tggtaaccgt ataaatttcc ctagttacgg ggtcttcaat 1020

cctggtggcg ccatttggat tgcagatgag gatccacgtc ctttttatcg gacattatca 1080

gatcctgttt ttgtccgagg aggatttggg aatcctcatt atgtactggg gcttagggga 1140

gtagcatttc aacaaactgg tacgaaccac acccgaacat ttagaaatag tgggaccata 1200

gattctctag atgaaatccc acctcaggat aatagtgggg caccttggaa tgattatagt 1260

catgtattaa atcatgttac atttgtacga tggccaggtg agatttcagg aagtgattca 1320

tggagagctc caatgttttc ttggacgcac cgtagtgcaa cccctacaaa tacaattgat 1380

ccggagagga ttactcaaat accattggta aaagcacata cacttcagtc aggtactact 1440

gttgtaagag ggcccgggtt tacgggagga gatattcttc gacgaacaag tggaggacca 1500

tttgcttata ctattgttaa tataaatggg caattacccc aaaggtatcg tgcaagaata 1560

cgctatgcct ctactacaaa tctaagaatt tacgtaacgg ttgcaggtga acggattttt 1620

gctggtcaat ttaacaaaac aatggatacc ggtgacccat taacattcca atcttttagt 1680

tacgcaacta ttaatacagc ttttacattc ccaatgagcc agagtagttt cacagtaggt 1740

gctgatactt ttagttcagg gaatgaagtt tatatagaca gatttgaatt gattccagtt 1800

actgcaacat ttgaagcaga atatgattta gaaagagcac aaaaggcggt gaatgcgctg 1860

tttacttcta taaaccaaat agggataaaa acagatgtga cggattatca tattgatcaa 1920

gtatccaatt tagtggattg tttatcagat gaattttgtc tggatgaaaa gcgagaattg 1980

tccgagaaag tcaaacatgc gaagcgactc agtgatgagc ggaatttact tcaagatcca 2040

aacttcaaag gcatcaatag gcaactagac cgtggttgga gaggaagtac ggatattacc 2100

atccaaagag gagatgacgt attcaaagaa aattatgtca cactaccagg tacctttgat 2160

gagtgctatc caacgtattt atatcaaaaa atagatgagt cgaaattaaa accctatact 2220

cgttatcaat taagagggta tatcgaggat agtcaagact tagaaatcta tttgatccgc 2280

tataatgcaa aacacgaaac agtaaatgtg ctaggtacgg gttctttatg gccgctttca 2340

gtccaaagtc caatcagaaa gtgtggagaa ccgaatcgat gcgcgccaca ccttgaatgg 2400

aatcctgatc tagattgttc ctgcagagac ggggaaaaat gtgcacatca ttcgcatcat 2460

ttctccttgg acattgatgt tggatgtaca gacttaaatg aggacttaga tgtatgggtg 2520

atattcaaga ttaagacgca agatggccat gcaagactag gaaatctaga gtttctcgaa 2580

gagaaaccat tagtcgggga agcactagct cgtgtgaaaa gagcagagaa aaaatggaga 2640

gataaacgtg aaaaattgga attggaaaca aatattgttt ataaagaggc aaaagaatct 2700

gtagatgctt tatttgtaaa ctctcaatat gatcaattac aagcggatac gaatattgcc 2760

atgattcatg cggcagataa acgtgttcat agaattcggg aagcgtatct tccagagtta 2820

tctgtgattc cgggtgtaaa tgtagacatt ttcgaagaat taaaagggcg tattttcact 2880

gcattcttcc tatatgatgc gagaaatgtc attaaaaacg gtgatttcaa taatggctta 2940

tcatgctgga acgtgaaagg gcatgtagat gtagaagaac aaaacaacca ccgttcggtc 3000

cttgttgttc cggaatggga agcagaagtg tcacaagaag ttcgtgtctg tccgggtcgt 3060

ggctatatcc ttcgtgtcac agcgtacaag gagggatatg gagaaggttg cgtaaccatt 3120

catgagatcg agaacaatac agacgaactg aagtttagca actgcgtaga agaggaagtc 3180

tatccaaaca acacggtaac gtgtaatgat tatactgcaa atcaagaaga atacgggggt 3240

gcgtacactt cccgtaatcg tggatatgac gaaacttatg gaagcaattc ttctgtacca 3300

gctgattatg cgtcagtcta tgaagaaaaa tcgtatacag atggacgaag agacaatcct 3360

tgtgaatcta acagaggata tggggattac acaccactac cagctggcta tgtgacaaaa 3420

gaattagagt acttcccaga aaccgataag gtatggattg agatcggaga aacggaagga 3480

acattcatcg tggacagcgt ggaattactc cttatggagg aatag 3525

<210>4

<211>1174

<212>PRT

<213> Artificial sequence

<220>

<223>

<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 Phe Glu Ala Glu Tyr

595 600 605

Asp Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Leu Phe Thr Ser Ile

610 615 620

Asn Gln Ile Gly Ile Lys Thr Asp Val Thr Asp Tyr His Ile Asp Gln

625 630 635 640

Val Ser Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cys Leu Asp Glu

645 650 655

Lys Arg Glu Leu Ser Glu Lys Val Lys His Ala Lys Arg Leu Ser Asp

660 665 670

Glu Arg Asn Leu Leu Gln Asp Pro Asn Phe Lys Gly Ile Asn Arg Gln

675 680 685

Leu Asp Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Ile Gln Arg Gly

690 695 700

Asp Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gly Thr Phe Asp

705 710 715 720

Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu Ser Lys Leu

725 730 735

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

740 745 750

Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys His Glu Thr Val

755 760 765

Asn Val Leu Gly Thr Gly Ser Leu Trp Pro Leu Ser Val Gln Ser Pro

770 775 780

Ile Arg Lys Cys Gly Glu Pro Asn Arg Cys Ala Pro His Leu Glu Trp

785 790 795 800

Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Lys Cys Ala His

805 810 815

His Ser His His Phe Ser Leu Asp Ile Asp Val Gly Cys Thr Asp Leu

820 825 830

Asn Glu Asp Leu Asp Val Trp Val Ile Phe Lys Ile Lys Thr Gln Asp

835 840 845

Gly His Ala Arg Leu Gly Asn Leu Glu Phe Leu Glu Glu Lys Pro Leu

850 855 860

Val Gly Glu Ala Leu Ala Arg Val Lys Arg Ala Glu Lys Lys Trp Arg

865 870 875 880

Asp Lys Arg Glu Lys Leu Glu Leu Glu Thr Asn Ile Val Tyr Lys Glu

885 890 895

Ala Lys Glu Ser Val Asp Ala Leu Phe Val Asn Ser Gln Tyr Asp Gln

900 905 910

Leu Gln Ala Asp Thr Asn Ile Ala Met Ile His Ala Ala Asp Lys Arg

915 920 925

Val His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Leu Ser Val Ile Pro

930 935 940

Gly Val Asn Val Asp Ile Phe Glu Glu Leu Lys Gly Arg Ile Phe Thr

945 950 955 960

Ala Phe Phe Leu Tyr Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe

965 970 975

Asn Asn Gly Leu Ser Cys Trp Asn Val Lys Gly His Val Asp Val Glu

980 985 990

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

995 1000 1005

Glu Val Ser Gln Glu Val Arg Val Cys Pro Gly Arg Gly Tyr Ile

1010 1015 1020

Leu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cys Val

1025 1030 1035

Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ser

1040 1045 1050

Asn Cys Val Glu Glu Glu Val Tyr Pro Asn Asn Thr Val Thr Cys

1055 1060 1065

Asn Asp Tyr Thr Ala Asn Gln Glu Glu Tyr Gly Gly Ala Tyr Thr

1070 1075 1080

Ser Arg Asn Arg Gly Tyr Asp Glu Thr Tyr Gly Ser Asn Ser Ser

1085 10901095

Val Pro Ala Asp Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr Thr

1100 1105 1110

Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg Gly Tyr Gly

1115 1120 1125

Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu Leu Glu

1130 1135 1140

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

1145 1150 1155

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

1160 1165 1170

Glu

<210>5

<211>2652

<212>DNA

<213> Artificial sequence

<220>

<223>

<400>5

agattacaag gtagtgaatt gtgacatgta ttcgttccta tccgatccgt cgtttttgag 60

cactaggtgc ggtcactgtg acgcgtggac ttggcttcgc ccactgccat cgtggaccca 120

cgtcatcagc aagtgtccat atccaccacc cgacccgacg accgcttgcc gtccgatccg 180

tgtgctcccg agggcaagga tggcatttcg ccacgcgaga tatttttcgg tggcctgcac 240

aggccggcag tgcagcggcc aaaacgaggt caggtcagtc acgctgggcc ccgcctcacg 300

ctcccgtcct gctccgggtcccaacaaagc cgtccccggg aggtgctcgt gtgctcgtag 360

cgcggtggcg accccgatgc cccgcatatt ccactgggcg tccgcgccgt cggatgggat 420

caggacggcc gcggcggccc cgcgctcggc tataaagacg ctgcggggga cgcattccct 480

ctccgtgctt tcttagaggt gggttggctt ctcctccccc tccggttcgg gttcgggttc 540

gtgaggttct ccggggttcg ggttcgtggg tgagcggatc gagactagta tggagaacaa 600

catccagaac cagtgcgtcc cctacaactg cctcaacaac cccgaggtcg agatcctcaa 660

cgaggagcgc tccaccggcc gcctccccct cgacatctcc ctctccctca cccgcttcct 720

cctctccgag ttcgtccccg gcgtcggcgt cgccttcggc ctcttcgacc tcatctgggg 780

cttcatcacc ccctccgact ggtccctctt cctcctccag atcgagcagc tcatcgagca 840

gcgcatcgag accctcgagc gcaaccgcgc catcaccacc ctccgcggcc tcgccgactc 900

ctacgagatt tacatcgagg ccctccgcga gtgggaggcc aaccccaaca acgcccagct 960

ccgcgaggac gtccgcatcc gcttcgccaa caccgacgac gccctcatca ccgccatcaa 1020

caacttcacc ctcacctcct tcgagatccc cctcctctcc gtctacgtcc aggccgccaa 1080

cctccacctc tccctcctcc gcgacgccgt ctccttcggc cagggctggg gcctcgacat 1140

cgccaccgtc aacaaccact acaaccgcct catcaacctc atccaccgct acaccaagca 1200

ctgcctcgac acctacaacc agggcctcga gaacctccgc ggcaccaaca cccgccagtg 1260

ggcccgcttc aaccagttcc gccgcgacct caccctcacc gtcctcgaca tcgtcgccct 1320

cttccccaac tacgacgtcc gcacctaccc catccagacc tcctcccagc tcacccgcga 1380

gatttacacc tcctccgtca tcgaggactc ccccgtctcc gccaacatcc ccaacggctt 1440

caaccgcgcc gagttcggcg tccgcccccc ccacctcatg gacttcatga actccctctt 1500

cgtcaccgcc gagaccgtcc gctcccagac cgtctggggc ggccacctcg tctcctcccg 1560

caacaccgcc ggcaaccgca tcaacttccc ctcctacggc gtcttcaacc ccggcggcgc 1620

catctggatc gccgacgagg acccccgccc cttctaccgc accctctccg accccgtctt 1680

cgtccgcggc ggcttcggca acccccacta cgtcctcggc ctccgcggcg tcgccttcca 1740

gcagaccggc accaaccaca cccgcacctt ccgcaactcc ggcaccatcg actccctcga 1800

cgagatcccc ccccaggaca actccggcgc cccctggaac gactactccc acgtcctcaa 1860

ccacgtcacc ttcgtccgct ggcccggcga gatttccggc tccgactcct ggcgcgcccc 1920

catgttctcc tggacccacc gctccgccac ccccaccaac accatcgacc ccgagcgcat 1980

cacccagatc cccctcgtca aggcccacac cctccagtcc ggcaccaccg tcgtccgcgg 2040

ccccggcttc accggcggcg acatcctccg ccgcacctcc ggcggcccct tcgcctacac 2100

catcgtcaac atcaacggcc agctccccca gcgctaccgc gcccgcatcc gctacgcctc 2160

caccaccaac ctccgcatct acgtcaccgt cgccggcgag cgcatcttcg ccggccagtt 2220

caacaagact atggacaccg gcgaccccct caccttccag tccttctcct acgccaccat 2280

caacaccgcc ttcaccttcc ccatgtccca gtcctccttc accgtcggcg ccgacacctt 2340

ctcctccggc aacgaggtct acatcgaccg cttcgagctg atccccgtct gaggtcaccc 2400

gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga 2460

ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga 2520

cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga 2580

tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt 2640

tactagatcg gg 2652

Claims

1. The nucleic acid molecule of the protein mCry1F is coded, and the nucleotide sequence of the nucleic acid molecule is shown as a sequence 1 in a sequence table.

2. An expression cassette, recombinant vector or recombinant microorganism comprising the nucleic acid molecule of claim 1.

3. Use of the nucleic acid molecule of claim 1 or the expression cassette, recombinant vector or recombinant microorganism of claim 2, e1) or e2) or e 3): e1) improving the insect resistance of the plants; e2) preparing a product with an insect-resistant effect; e3) cultivating a transgenic plant with improved insect resistance; the plant is corn; the insect resistance is against Asiatic corn borer.

4. Use according to claim 3, characterized in that: the product is a medicament.

5. A method of breeding a transgenic plant comprising the steps of: introducing the nucleic acid molecule of claim 1 into a recipient plant to produce a transgenic plant; (ii) an increased insect resistance of the transgenic plant compared to the recipient plant; the plant is corn; the insect resistance is against Asiatic corn borer.

6. A method of plant breeding comprising the steps of: increasing the level of mCry1F protein encoded by the nucleic acid molecule of claim 1 in a plant, thereby increasing insect resistance; the plant is corn; the insect resistance is against Asiatic corn borer.