CN101113424B - Coleoptera pest efficient Bacillus thuringiensis cry8G gene, protein and uses thereof - Google Patents

Abstract

The invention relates to "bacillus thuringiensis cry8G gene, protein and application thereof with high efficiency against coleopteran pests", belonging to the technical field of biological control. The present invention provides the nucleotide sequence of the Bacillus thuringiensis cry8G gene with high toxicity to coleopteran pests and the amino acid sequence of the protein encoded by it, as well as the nucleotide sequence and the amino acid sequence of the gene artificially designed for transgenic plants. encoded amino acid sequence. By using the above-mentioned genes or their artificially designed sequences to transform microorganisms and plants, they can show toxicity to related pests, and overcome or delay insects' resistance to engineering bacteria and transgenic plants.

Description

Bacillus thuringiensis cry8G gene, protein and application thereof highly effective against coleopteran pests

technical field

The invention belongs to the technical field of biological control. The invention relates to the nucleotide sequence of the cry8G gene highly toxic to the Coleopteran pests, to the amino acid sequence of the protein highly toxic to the Coleopteran pests, and to artificially designed and synthesized cry8G genes that can be used in plants. The nucleotide sequence of the cry8G gene expressed in , relates to the amino acid sequence of the protein encoded by the nucleotide sequence of the artificially designed cr8G gene, relates to a recombinant strain containing the cry8G gene, relates to using the gene to construct an expression vector, and also relates to using the above-mentioned gene Sequences for methods of plant transformation. the

Background technique

Scarab belongs to Coleoptera Scarabaeidae (Scarabaeidae), and its larvae (commonly known as grubs, also referred to as "grubs" for short in the present invention hereinafter) are a class of important worldwide distribution underground pests, which can harm food, cotton, oil crops, vegetables, sugar Forage crops, tobacco, grass, flowers, lawn grass, fruit trees and other plants. A large number of surveys have shown that grubs are the most harmful underground pests, among which mainly the larvae of the family Beetleidae and Beetleidae, accounting for more than 70-80% of the total underground pests. According to statistics, the occurrence area of grubs in the whole country is about 100 million mu every year, and it once reached 320 million mu in severe years. The yield loss is as high as more than 20%, and some plots even go out of production. In recent years, the Huanghuaihai region has the largest occurrence area and the largest number of occurrences, mainly harming crops such as grain and oil crops; other regions are also very serious. For example, grubs that harm sugarcane are common in Guangdong, Guangxi, Yunnan, Sichuan, Fujian and other places. in Tibet, Qinghai, Gansu, Xinjiang and other western regions, the occurrence of grubs is also very serious (Wei Hongjun et al., "China's Subterranean Pests", Shanghai: Shanghai Science and Technology Press, 1989, 1-41; Wang Yongxiang et al., "Jizhong Species of grubs in plain areas and comprehensive control techniques", "Journal of Hebei Normal University" (Natural Science Edition), 1998, 22(2): 268-270). Take peanut as an example, which ranks second only to rapeseed in the planting area of my country's oil crops. my country's peanut production accounts for about 35% of the world's total peanut production, ranking first in the world, with an annual export revenue of 20.7 billion U.S. dollars. In 2001, the national peanut area (5 million hectares) and total production (14.5 million tons) both reached the highest level in history. But the harm of grubs to peanuts is very serious. In order to control the harm of grubs, comprehensive control strategies such as agriculture, chemistry, and physics are generally adopted. Although this has certain effects, it is difficult to achieve the effect of continuous control. Therefore, finding new and effective methods of prevention and treatment has become a top priority. the

On the basis of obtaining the Bt gene with high toxicity to grubs, breeding grub-killing transgenic plants is a new prevention and control approach worth exploring. the

Bacillus thuringiensis (Bt) is a Gram-positive bacterium with a wide distribution. While forming spores, it can produce parasporal crystals of protein nature, which are suitable for Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera Various insects such as Homoptera, Orthoptera, and Mallophaga, as well as nematodes, mites and protozoa have specific insecticidal activity (Schnepf, E.N.et al, Microbiol.And Molecular Biology Review , 1998, 62:3775-806). This insecticidal crystal protein (Insecticidal Crystal Proteins, ICPs), also known as δ-endotoxin (delta-endotoxin), is harmless to humans and animals, and does not pollute the environment, so Bt has been the most widely used in the biological control of pests. the

At present, nearly 400 Bt insecticidal genes encoding insecticidal crystal proteins have been cloned, and they belong to 157 model genes. In recent years, the international research trends of cry8 genes have attracted attention. Studies have shown that this type of gene has insecticidal effects on various Coleoptera pests such as scarabs, weevils, and phyllids. In 1992, Ohba et al. screened out a new strain (B.t.subsp. Japonensis BuiBui) with specific insecticidal activity to scarab larvae from Bt strains for the first time in the world (Ohba, M.et al., A unique isolate of Bacillus thuringiensis serovar japonensis with a highlarvicidal activity specific for scarabaeid beetles, Letters in Applied Microbiology, 1992.14:54-57), in 1994 Sato etc. cloned a kind of new insecticidal gene cry8C from it (Sato, R.et al, Cloning, heterologous expression, and localization of a novel crystal protein gene from Bacillus thuringiensis serovar japonensis strain buibui toxic to scarabaeidinsects, Curr. Microbiol. 1994.28: 15-19.4). At present, 11 kinds of cry8 genes have been found, and the encoded protein consists of 1160-1210 amino acids, and the molecular weight is between 128-137kDa. See Table 1 for detailed information (Asano, S., Yamanaka, S. and Takeuchi, K., Protein having insecticidal activity, DNA encoding the protein, and controlling agent and controlling method of noxious organisms, 2002, JP 2002045186-A and JP 20062045 -A/2)). Among them, Cry8Aa1 and Cry8Ba1 separated by Mycogen Company of the United States have obvious insecticidal activity against various pests of the scarab family (Tracy E. Michaels, et al., Bacillusthuringiensis toxins active against scarab pests, 1994, USP5554534). The U.S. isolated two genes cry8Bb1 and cry8Bc1 genes from the Bt strain, and found that they have significant insecticidal effects on Western cornrootworm and have been used for the development of transgenic insect-resistant corn (Abad, Andre, R., DuckNicholas, B., Feng, Xiang, Flannagan Ronald, D., Kahn, Theodore, W., Sims, Lynne, E. Genes encoding novel proteins with pesticide activity against coleopterans, 2002, WO 02/34774A2). In my country, the Institute of Plant Protection of Hebei Academy of Agricultural Sciences and Hebei Agricultural University have successively screened and obtained a number of Bt strains with specific insecticidal activity against the larvae of Anomala exoleta and A. corpulenta. The indoor bioassay mortality rate reached 100% (Feng Shuliang et al., "A new isolate of Bacillus thuringiensis with insecticidal activity to scarab larvae", "China Biological Control", 2000, 16 (2): 74-78). the

Table 1 Bacillus thuringiensis Cry8 insecticidal crystal protein

Contents of the invention

The present invention provides a Bacillus thuringiensis cry8G model gene sequence with high toxicity to important Coleopteran pests such as the black gill beetle, which can be applied to the transformation of microorganisms and plants, so that it can show toxicity to related pests, and overcome and delay Resistance of pests to engineered bacteria and transgenic plants. the

Bacillus thuringiensis strain HBF-18, its preservation number is CGMCC2070. the

The nucleotide sequence of the Bacillus thuringiensis cry8Ga1 gene highly effective against coleopteran pests is shown in SEQ ID NO1. the

An engineering bacteria strain BioT8G is characterized in that it contains a cry8Gal gene. the

The Bacillus thuringiensis cry8Ga1 protein highly effective against coleopteran pests is encoded by the above cry8Ga1 gene, and its amino acid sequence is shown in SEQ ID NO2. the

Application of cry8Ga1 protein in preparation of medicament for killing coleopteran pests. the

A protein that has the same function as the above-mentioned protein, and its amino acid sequence is shown in SEQ ID NO4. 

An artificially modified and synthesized mcry8Ga1 gene encodes the above-mentioned protein, and its nucleotide sequence is shown in SEQ ID NO3. the

A plant expression vector pBSmGN, characterized in that the plant expression vector consists of mcry8Ga1 gene sequence, a constitutive expression promoter or a root-specific promoter, a terminator and a binary cell that can shuttle between Escherichia coli and Agrobacterium tumefaciens The carrier is constructed. the

Application of mcry8Ga1 gene in plant resistance to coleopteran pests. the

The application is to transform plants or microorganisms with the plant expression vector pBSmGN containing the mcry8Gal gene, so as to produce toxicity against coleopteran pests. the

The plant is tobacco. the

The application is to prepare the protein expressed by the mcry8Gal gene into a medicament for killing the coleopteran pests. the

The present invention isolates and obtains strain HBF-18 from soil in Hebei, and its preservation number is CGMCC2070, and its biological characteristic is that it can produce spores during the growth cycle, and at the same time produce toxic parasporal crystals. the

A pair of universal primers were designed according to the conserved region of the cry8 gene:

SN5un85`-GTCCGAATAATCAGAATGAATATG-3`

SN3un85`-CGTTTCGCCTCTCTCACTGCAT-3`

PCR amplification identified the HBF-18 bacterial strain, the amplification result (see accompanying drawing 1), and its display band is all different from known cry8 class gene (see table 3), shows that may contain new cry8 insecticidal in the bacterial strain HBF-18 Gene. the

A pair of full-length gene primers cry8G5/cry8G3 was designed to amplify the full-length gene. And introduce BamHI/SalI for cloning and expression, the sequence of the primer pair cry8G5/cry8G3 is as follows:

BamHI

cry8G5: 5′-CGC GGATCC GAAATGAGTCCGAATAATCA-3′

SalI

cry8G3: 5′-ACGC GTCGAC CTCTTTCTTCTAACACGAGT-3′

Using the total DNA of bacterial strain HBF-18 as a template, PCR amplification was performed with pfuDNA polymerase, and the result (see Figure 2) showed that a 3.5Kb band was amplified, which was connected with the vector pET21b to transform Escherichia coli JM110 to obtain recombinant Plasmid pSAS018 (Fig. 2). Sequencing analysis was performed on the inserted fragment, and the obtained sequence SEQ ID NO 1 was a BamHI/SalI double-digested fragment in pSAS018, with a full length of 3472bps. The analysis showed that it contained an open reading frame, the ORF1 position was 1-3472, and the GC content was 38. %, encoding a protein consisting of 1157 amino acids. After determination, its amino acid sequence is shown in SEQ ID NO 2. Homology analysis showed that this protein has high homology with Cry8 proteins, and Table 4 shows its homology data. Since the amino acid homology with known Cry8 proteins is lower than 78%, the highest is only 58.2% (Cry8Bb1), it was named Cry8Ga1 by the Bt insecticidal crystal protein nomenclature committee. the

Primers cry8G5/cry8G3 were introduced into the BamHI and SalI sites respectively, using the plasmid DNA of the strain HBF-18 as a template, the full-length gene was amplified, inserted into the Bt expression vector pSTK to obtain the recombinant plasmid pSK018 (see Figure 3), and transformed into Escherichia coli SCS110 , extract the plasmid, and transform the Bt crystal-free mutant strain HD-73 ^- zhong by electric shock (this mutant strain comes from the Biotechnology Laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, and can be provided to the public, see Li Haitao et al., Journal of Agricultural Biotechnology 2005Vol.13No .6P.787-791), to obtain engineering bacteria BioT8G.

The above engineering bacteria BioT8G were cultured at 30°C in beef extract medium (5 grams of peptone, 3 grams of beef extract, 10 grams of glucose, 1000 mL of water, 121°C, 20 minutes of high-pressure steam sterilization), and the protein was extracted for SDS-PAGE electrophoresis Analysis (method is referring to Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 1989), result (see accompanying drawing 4). The results showed that the cry8Ga1 gene in the engineering bacteria Biot8G was expressed, and the molecular weight of the expressed product was about 130kDa. the

The activity determination of Cry8Ga1 protein showed that the expressed Cry8Ga1 had the activity of killing the larvae of North China giant beetle and black beetle. the

The 1-2040bp sequence of the cry8Gal gene was optimized according to the difference in codon preference between microorganisms and plants. The present invention carries out whole gene synthesis according to the artificially modified sequence of cry8Ga1 gene, the nucleotide sequence shown in SEQ ID NO 3 for the new gene, and the corresponding protein sequence shown in SEQ ID NO 4. The nucleotide sequence homology between the cry8Ga1 gene and the mcry8Ga1 (modified cry8Ca1) gene is only 86.88%, and the G+C content is also increased from 37.6% of the original cry8Ca1 to 45.2%. The amino acid codon usage frequency of the Cry8Ga1 protein was adjusted so that the amino acid codon usage frequency of the mCry8Ga1 protein was close to the usage frequency in plants, and both ends of the artificially modified sequence of the cry8Ca gene were introduced into BamHI, KpnI, and SacI sites (see SEQ ID NO 3), connected to the pUC57 vector (this vector is a commonly used vector, GenBank accession number is Y14837), and the recombinant plasmid was named pUC57-mcry8G. the

When artificially synthesizing the transformed Bt cry8G gene, digest the plasmid pUC57-mcry8G with BamHI and SacI to reclaim the 2.0kb fragment, and use the same endonuclease to digest the plasmid pBI121 (this vector is a commonly used vector, and the GenBank accession number is AF485783. See Chen PY, et al, 2003, Mol.Breed, 11: 287-293), recovered the 12kb fragment, connected the two fragments, transformed JM110, and obtained a positive transformant. The newly constructed plasmid was named pBSmGN. The plasmid contains a constitutive expression promoter CaMV35S (that is, a DNA sequence that can drive the connected gene fragments to be transcribed, and then translated into proteins. The constitutively expressed promoter can regulate genes at any stage of growth and development and in any tissue. expression), the mcry8Gal gene, and the NOS terminator (a DNA sequence that contains a signal to terminate gene expression). The construction diagram of the plasmid is shown in the accompanying drawing 5, and the plasmid can be transformed into a plant to obtain a transgenic plant. the

Transform the artificially modified gene mcry8Ga1 Agrobacterium to prepare positive clones, and then transform tobacco. The bioactivity test of the transgenic tobacco shows that the transgenic plants show good resistance to Holotrichia parallela. the

Tobacco is a commonly used model plant for verifying gene functions in plant genetic engineering. The anti-Holotrichia parallela performance of transgenic tobacco is due to the fact that there is a promoter in the plant to initiate the transcription of the artificially modified mcry8Ga1 gene, which expresses the Cry8G protein. The expression cassette in the meta-vector - constitutive promoter, artificially modified mcry8Ga1 and terminator can only express foreign genes when integrated into the tobacco genome, so any plant with established Agrobacterium transformation methods can be transformed with this binary vector , the obtained transgenic plants all have the property of resisting the black beetle (Holotrichia parallela). the

Biological deposit information:

Microorganism name (scientific name of genus and species): Bacillus thuringiensis

Preservation unit: General Microbiology Center of China Microbiological Culture Collection Management Committee

Address of Preservation Unit: No. 13, North 1st Street, Zhongguancun, Haidian District, Beijing

Description of drawings

Date of preservation: June 1, 2007

Deposit number: CGMCC No.2070

Figure 1: PCR-RFLP profile of strain HBF-18. in:

M.DNA Molecular Weight Standard

1. Enzyme digestion of PCR products

Figure 2: Restriction map of recombinant plasmid pSAS018. in:

M.DNA Molecular Weight Standard

1. PCR product BamHI/SalI digestion

2. Carrier pET21bBamHI/SalI digestion

3. Restriction map of recombinant plasmid pSAS018BamHI/SalI

Figure 3: Restriction map of recombinant plasmid pSK018. in:

M.DNA Molecular Weight Standard

1. Carrier pSTK BamHI/SalI digestion

2. PCR product BamHI/SalI digestion

3. Restriction map of recombinant plasmid pSK018BamHI/SalI

Figure 4: Expression of cry8Ga1 gene in Bt amorphous mutant. in:

M. Protein Molecular Weight Standards

1. HBF-18

2. Biot8G

3.HD- ^73-

Figure 5: Construction diagram of pBSmGN plasmid

Figure 6: Molecular detection of transformed tobacco, where:

M. Protein Molecular Weight Standards

1, 2. The detection results of some positive transgenic plants

Detailed ways

The present invention will be described in further detail below in conjunction with embodiment. the

Example 1

1.1 Identification of cry gene in strain 185

A pair of universal primers were designed according to the conserved region of the cry8 gene

SN5un85`-GTCCGAATAATCAGAATGAATATG-3`

SN3un85`-CGTTTCGCCTCTCTCACTGCAT-3`

Table 2 shows the homologous sequences of these genes and primers, and Table 3 shows the size of the digested fragments of cry8 gene amplified products predicted by this pair of primers. These genes can be identified respectively by this PCR-RFLP method. the

Table 2 The pairing situation of the primers and the conserved regions of cry8 genes and the position of the paired regions on the gene

Table 3 PCR amplification products and restriction enzyme length polymorphisms of cry8

Bt strain 185 was identified with the following PCR reaction system (50 μL):

10×PCR buffer 5μL

_MgCl2 (20mM) 6μL

dNTP(10mM) 1μL

Primer pair (10mM) 1μL/each

Template 1uL

Taq polymerase (5U/μL) 0.5μL

Make up to 50 μL with ultrapure water, mix well and centrifuge, add 30 μL of paraffin oil. the

Amplification cycle: denaturation at 94°C for 1 minute, annealing at 54°C for 1 minute, extension at 72°C for 4 minutes, 25 cycles, and finally extension at 72°C for 10 minutes. the

Utilize KpnI and DraI digestion analysis to the PCR product, the result (accompanying drawing 1) shows that the band is 1260bp, 570bp, 320bp, is different from the map of known cry8 class gene (table 3), shows that bacterial strain HBF-18 may contain new cry8 insecticidal gene.

1.2 Cloning of cry8G gene in strain HBF-18

A pair of full-length gene primers cry8G5/cry8G3 was designed to amplify the full-length gene. And introduce BamHI/SalI for cloning and expression, the sequence of the primer pair cry8G5/cry8G3 is as follows:

BamHI

cry8G5: 5′-CGC GGATCC GAAATGAGTCCGAATAATCA-3′

SalI

cry8G3: 5′-ACGC GTCGAC CTCTTTCTTCTAACACGAGT-3′

Using the total DNA of strain HBF-18 as a template, pfuDNA polymerase was used to perform PCR amplification with the following system. . the

10×PCR buffer 5μL dNTP (10mM) 1μL Primer pair (10mM) 1μL/piece template 1uL pfuDNA polymerase (5U/μL) 0.5μL

Make up to 50 μL with ultrapure water, mix well and centrifuge, add 30 μL of paraffin oil. the

Amplification cycle: denaturation at 94°C for 1 minute, annealing at 54°C for 1 minute, extension at 72°C for 4 minutes, 25 cycles, and finally extension at 72°C for 10 minutes. The results (see accompanying drawing 2) showed that a 3.5Kb band was amplified, which was connected with the vector pET21b to transform Escherichia coli JM110, and a positive transformant pSAS018 was obtained. Sequencing analysis was performed on the inserted fragment, and the obtained sequence SEQ ID NO 1 was a BamHI/SalI double-digested fragment in pSAS018, with a full length of 3472bps. The analysis showed that it contained an open reading frame, the ORF1 position was 1-3472, and the GC content was 38. %, encoding a protein consisting of 1157 amino acids. After determination, its amino acid sequence is shown in SEQID NO 2. Homology analysis showed that this protein has high homology with Cry8 proteins, and Table 4 shows the homology data. Since the amino acid homology with known Cry8 proteins is lower than 78%, the highest is only 58.2% (Cry8Bb1), it was named Cry8Ga1 by the Bt insecticidal crystal protein nomenclature committee. the

Table 4 Homologous comparison data of Cry8Ga1 and Cry8 proteins

The present invention further analyzes the amino acid composition of the Cry8Ga1 protein (see Table 6), and learns that its molecular weight is 131.56kDa, and the isoelectric point is pH4.735 (see Table 5), and analyzes the biochemical index of the protein (see Table 5)

Table 5 Biochemical properties of Cry8Ga1 protein

Amino acid composition of table 6Cry8Ga1 protein

1.3 expression of cry8G gene

Primers cry8G5/cry8G3 were introduced into the BamHI and SalI sites respectively, and the full-length gene was amplified by using the plasmid DNA of the strain HBF-18 containing the full-length cry8Ga1 as a template, inserted into the Bt expression vector pSTK (see Figure 3), and transformed into Escherichia coli SCS110, extract the plasmid, and transform the Bt crystal-free mutant strain HD- ^73- by electroporation to obtain the engineering bacterium BioT8G.

Cultivate the above two strains of engineering bacteria in beef extract medium at 30°C for 30 hours, respectively, take 500 μL of the bacterial solution into an Eppendorf tube, and ultrasonically break for 30 seconds (B. Braun U Labsonic, 230V, T _interval = 0.5 seconds); Add 25 μL freshly prepared 0.5N NaOH to 100 μL, and react at 25°C for 5 minutes; add 65 μL 3×sample buffer (925 μL sample buffer + 75 μL β-mercaptoethanol), and boil at 100°C for 5 minutes. The precipitate was removed by centrifugation. 10uL of the sample was loaded for SDS-PAGE electrophoresis to analyze the results. The results (Fig. 4) show that all the cry8Ga1 genes in the engineering bacteria Biot8G are expressed, and the molecular weight of the expressed product is about 130 kDa.

1.4 Activity determination of Cry8G protein

Bt engineered strains were inoculated on common bacterial agar Kirschner flask medium and cultured for 3 days. The recipient strain HD- ^73- was inoculated on common bacterial agar Kelvin flask medium and cultured for 4 days. Wash the culture, dilute it with 2-fold gradient concentration, add 40ml of the bacterial suspension to 200g of fine soil (sterilized by ultraviolet light) with evenly thick and thin potato shreds, mix well, and keep the soil water content at 18%-20%. Inject 20 15-day-old larvae of the black beetle, take the treatment of adding water as the blank control, infect and raise at 28°C, check the number of dead insects on day 14, and calculate LC ₅₀ . The results (see Table 8) show that the engineered strains have extremely high poisonous activity against the North China giant black beetle (Holotrichiaoblita) and the dark beetle (Holotrichia parallela). The Cry8G protein expressed by it has the activity of killing the North China giant black beetle and the dark beetle.

Table 7 Bt engineering bacteria and HBF-18 bacterial strain are measured to the insecticidal activity of North China's large black beetle (Holotrichia oblita), dark black beetle (Holotrichia parallela) larvae

Note: In the concentration unit of the result, the conversion of ×10 ⁸ /g soil and ×10 ⁸ /ml is to add 36ml of bacterial suspension to every 200g of soil.

Example 2

2.1 Nucleotide sequence of artificially designed and synthesized cry8G gene that can be expressed in plants

The 1-2040bp sequence of the cry8Gal gene was optimized according to the difference in codon preference between microorganisms and plants. The present invention carries out whole gene synthesis according to the artificially modified sequence of the cry8Ga1 gene, and the new gene is shown in the nucleotide sequence shown in SEQ ID NO 3. The nucleotide sequence homology between the cry8Ga1 gene and the mcry8Ga1 (modified cry8Ca1) gene was only 86.88%, and the G+C content was also increased from 37.6% of the original cry8Ca1 to 45.2% (Table 8). The amino acid codon usage frequency of the Cry8Gal protein was adjusted so that the amino acid codon usage frequency of the mCry8Gal protein was close to the usage frequency in plants (Table 9). The two ends of the artificially modified sequence of the cry8Ca gene were introduced into BamHI and KpnI sites, connected to the pUC57 vector, and the recombinant plasmid was named pUC57-mcry8G. the

Table 8 Comparison of the content of cry8Ga1 gene and mcry8Ga1G+C and the signal sequence of polyadenylation

The amino acid codon usage frequency of proteins in Table 9 plants, Cry8a1 and mCry8Ga1

2.2 Construction of plant expression vectors

Use BamHI and SacI to digest plasmid pUC57-mcry8G (preserved by the Biotechnology Group, Institute of Plant Protection, Chinese Academy of Agricultural Sciences) to recover a 2.0kb fragment, and use the same endonuclease to digest plasmid pBI121 (this vector is a commonly used vector, and the GenBank accession number is AF485783 See Chen PY, et al, 2003, Mol.Breed, 11: 287-293), recover the 12kb fragment, connect the two fragments, transform JM110, and obtain a positive transformant. The newly constructed plasmid is named pBSmGN. The plasmid contains a constitutively expressed promoter CaMV35S (that is, a DNA sequence that can drive the connected gene fragments to be transcribed, and then translated into protein. The constitutively expressed promoter can regulate the gene at any stage of growth and development and in any tissue. expression), the mcry8Ga gene and the NOS terminator (a DNA sequence that contains the termination signal for gene expression). See accompanying drawing 5 for the construction diagram of the plasmid, which can express foreign genes in plants. the

2.3 Agrobacterium transformation

Take a tube of 200 μl competent cells of Agrobacterium LBA4404, add 1 μg pBSmGN plasmid DNA, quick freeze in liquid nitrogen for 1 minute, resume culture at 37°C for 5 minutes, add 1ml YEB liquid medium, shake slowly (<100rpm) at 28°C for 4 hours , centrifuge at 1,000rpm for 30 seconds, discard the supernatant, add 100μl YEB liquid medium to resuspend the cells, spread on the YEB medium plate containing kanamycin 100μg/ml and streptomycin 125μg/ml, 28℃ Incubate for 48 hours. The clones on the YEB resistant medium plate were shaken, the plasmid was extracted, and the positive clones were detected by PCR method. the

2.4 Tobacco Transformation

Inoculate the Agrobacterium clone containing the pBSmGN plasmid in YEB liquid medium containing kanamycin 100 μg/ml and streptomycin 125 μg/ml, shake culture at 28°C until the OD ₆₀₀ is 0.6-0.8, and inoculate 1/50 in MS salt (pH7.0); cut the sterile tobacco seedlings into small pieces of 0.4×0.6cm ² , soak the tobacco leaves in MS salt, and infect with Agrobacterium for 10 minutes; take out the tobacco leaves, and dry the bacteria with sterile filter paper. solution, placed in MS medium covered with a layer of filter paper, cultured in the dark at 28°C for 3 days, and after 3 days, the tobacco leaves were transferred to MS selection differentiation medium (MS medium + 100 μg/ml kanamycin + 500 μg/ml carboxylate benzylmycin+3mg/ml 6-BA+0.2mg/ml NAA), 28°C, light/dark=16 hours/8 hours, after 2 weeks, green callus appeared on the edge of the leaf, and after 1 week, the callus The wounds were differentiated into small plants, and the small plants were excised and moved to the rooting medium (MS medium + 100 μg/ml kanamycin + 500 μg/ml carbenicillin) to take root. Continue to grow in the soil (ordinary soil: nutrient soil: vermiculite = 2:1:1).

2.5 Molecular detection of transformed tobacco

Extract the genomic DNA of transformed tobacco, take 1 μg of genomic DNA as a template, and the primer sequences are as follows:

8GF1: 5′-TTCAGTTGTCCACTCCGCCTA

8GR1: 5′-GCCATTCACAGCCTTCTTTGC

The reaction conditions of PCR amplification are: 94°C, 5 minutes, 1 cycle; 94°C, 1 minute, 53°C, 1 minute, 72°C, 2 minutes, 30 cycles. Electrophoresis the product. As shown in Figure 6, a fragment of 640 bp was amplified from the positive transformant. the

2.6 Bioactivity detection of transgenic tobacco

Two kinds of scarab adult populations, Holotrichia oblita and Holotrichia parallela, which are seriously harmful in northern my country, were collected from the field, brought back to the room, and put into breeding boxes of 40×40×50 cm respectively. Put 5-8cm thick damp sieved fine soil at the bottom, feed fresh elm leaves, and raise them at 26-28°C. After it lays eggs, the eggs are picked out and placed in moist soil to hatch. The newly hatched larvae were picked out, and 5 larvae were placed in each small rearing box (Φ8cm, H5cm), fed with potato pieces and fresh corn tender roots, and used for biotesting after the larvae grew to a certain age. the

The method of group planting and group inoculation was used for bioassay. The planting methods of planting transgenic plants and non-transformed plants separately or mixed planting were adopted in field soil, and 12 plants were treated for each treatment. the

According to the established harm degree grading standard, the harm degree and harm index statistics of North China giant black beetle (Holotrichia oblita) larvae to the transgenic plants tested are as follows, see Table 10. the

Table 10. Hazard statistics of transgenic plants and non-transgenic plants. the

From the statistical results in Table 10, the damage rate of the transgenic pBSmGN plants was 62.5%, and the damage index was 22.9; the damage rate of the non-transgenic plants was 100%, and the damage index was 72.9. The transgenic pBSmGN plants showed good resistance to North China black beetle (Holo trichia oblita). the

The damage degree of the larvae of the black gill beetle (Holotrichia parallela) to the transgenic plants tested is as follows. The damage rate of the transgenic pBSmGN plants was 50%, and the damage index was 12.5, which was lower than that of the non-transgenic plants, which was 100%, and the damage index of the untransformed plants was 75.0, which was equivalent. It can be seen that the transgenic pBSmGN plants showed good resistance to Holotrichia parallela. the

According to the above information, the gene can be expressed in the root of the plant by using the promoter that can specifically transcribe the gene in the root, so that the resistance to the target pest Holotrichia parallela can be obtained only in the root of the plant. the

Appendix: DNA sequence and protein sequence involved in the present invention

SEQ ID NO 1 (nucleotide sequence of cry8Ga1 gene)

      

TCCGA ATAATCAGAA CGAATATGAA ATTATAGATG CGTCATCACC TACTTCTGTA 1

TCCGA ATAATCAGAA CGAATATGAA ATTATAGATG CGTCATCACC TACTTCTGTA

61 TCTAATAACT CAGTGAAATA CCCTTTAGCA AGTGATCAAAA CGACCACATT ACAAAATATG

121 AACTATAAAG ATTATCTGAG AATGTCTGAG GGAGAGAATC CTGAATTATT TGGAAATCCA

181 GAGACGTTTA TTAGTGCGCA GGATGCGGTT GGAACTGGGA TTGATATTGT GAGTAAACTA

241 CTAGGTAGTT TAGGGGTTCC ACTTGTTGGG CAAGCCGCAA CGGCACTTAA ATGGATTATA

301 GGTAAATTGT GGCCTTCTTC AGGAAACCCG TGGGATGATT TGATGACGGC AGTAGAAGAA

361 CTCATAAATC AAAAAATAGA AGCATATGCA AGAAGTAAGG CACTTGCTGA ATTGGGTGTT

421 TCGGGAAGAG CTGTAAAATC CTATCAAACC GCACTTGAAG AGTGGCAAAA AAACCCGAAT

481 AACGCGCGAA GCGCAGCACT TGTAAGGGAA AGATTTTCAG ATGCAGAACA TACATTGCGT

541 ACTCAAATGA GTTTATTTAC CGTTCGTGGT TATGAAATTC CGCTTTTAGC AACATATGCA

601 CAAGCTGCCA ATTTGCATTT GTTTGTAATG AAGGATATTC AAATTTACGG GAGAGAATGG

661 GGATATACTC AGGGAGATAT TAACCTTTTC TATCGAGAAC AAGTAGAATT TACAGGGGAA

721 TACTCTGATT ATTGTGTTAA GTGGTACAAT GCTGGCTTAG ATAAATTAAG AGGCTCGACT

781 GCTCTACAAT GGATTAACTA TAATCGTTTC CGCAGAGAAA TGACAGTGAT GGCACTGGAT

841 ATAGTTGCAT TATTCCCAAA TTATGACATA CGCATGTATC CAATGAAAAC AACCGCAGAA

901 TTAACGCGAA GAATTTATAC AGATCCGCTT GGTTATACGG GAAGTGGGTC TAACACGCCA

961 CCATGGTATA ATTATGGATA TTCTTTCTCA TGGATAGAAA ATAATGCCGT GCCAGCACCT

1021 GGATTGTTCC AGTGGTTACA AGGAATTGGG ATTTATACTA AATTTGCTCG TATAACTCCA

1081 TTTTATGCGA ACTATTGGTC AGGACATACT GTATTTTATA AATTTACTAA CGATTCTACT

1141 GAGAGACGTG TTCAGTATGG AGATACAGAT ACTCCAGAAT TAGATAGTTC TTCCTTTGAA

1201 AATGTTGACA TTTATAAGGT TTCAGCATCA GTTGGTTCGT ACAAAAGTAA TACCGTACTA

1261 TTACCAACTT TTAAAGCTAC TTTTGAGGGG GTAAATCAAA ATAATCAGTT AAAGACCTTT

1321 AGGTATCAAA AAGAATCTAA TGTCCCAAGT CAAACGAAAA ACTCAACCAC AGAGCTGCCT

1381 GTTCAGTTAT CAACTCCGCC TACTTACGGA GATTCTGAAC AGTACAGTCA TAGACTAGCC

1441 TATGTTTTTG ATGCCCCAAT CGATTCATAT ACAGGCATAT ATCGCATGTA TGGATTTGCC

1501 CCTATTCTTG GTTGGACACA TATTAGTGTA AGTCGTGACA ATAGGATTGA TCCAGATAAA

1561 ATTACTCAAA TTCCAGCTGT AAAGGCATAT GCTGAGGGTC TTGCTAATTA TATCAAAGAT

1621 CCGGGGTTTA CAGGAGGAGA TTTATTAGCT TTAGGTAGAA ACTCAAATAC TTCATTGATT

1681 GTCAATTTTT CGAAGCCTCA AACATACCGT ATTCGTATTC GTTATGCTGC TAGTAAAACT

1741 TCGTATTTTC AACTACGTGG GCTGCATAAT ATAGCTCAGT CTCAGCGTTT CGAAGCGACG

1801 TATTCTAATA AAAATGAAAA CGATTTGACA TTTAACGATT TTAAATATGT AGAAATTCAA

1861 AAAACTGTTT CAATAGACAA TCCATCAGAA AGTCGTAGTA TAAGTATATA CACTCAATCA

1921 GATACAGAAT ACTTATTATG GACAAATCGA ATTCATCCCA GTAGATGCAA CATTTGGAGC

1981 GGAACAAGAC CTAGATGTGG CAAAGAAAGC GGTGAATGGC TTGTGTACCA ATACAAAAGA

2041 TGCCTTACAG ACAAGTGTAA CGGATTATCA AGTCAATCAA GCGGCAAACT TAGTAGAATG

2101 CCTATCGATG AGTTATACCC AAATGAAAAA CGCATGTTAT GGGATGCAGT GAAAGAGGCG

2161 AAACGACTTG TTCAGGCACG TAACTTACTC CAAGATACAG GCTTTAATGT AATAAATGGA

2221 GAAAACGGAT GGACGGGAAG TACGGGAATT GAGGTTGTGG AAGGGGATGT TCTGTTTAAA

2281 GATCGTTCGC TTCGTTTGCC AAGTGCGAGA GAGATTGATA CAGAAACATA TCCAACGTAT

2341 CTCTATCAAC AAATAGATGA ATCGCTTTTA AAACCATATA CAAGATATAG ACTAAGAGGT

2401 TTTATAGGAA GTAGTCAAGA TTTAGAGATT AAATTAATAC GTCATCGGGC AAATCAAATT

2461 GTCAAAAATG TACCGGATAA CCTCTTGCCA GATGTACGCC CTGTCAATTC TTGTGGTGGA

2521 GTCGATCGCT GCAGTGAACA ACAGTATGTA GACGCGAATT TAGCACTCGA AAACAATGGA

2581 GAAAATAGAA ATATGTCTTC TGATTCCCAT GCATTTTCTT TCCATATGGA TACAGGTGAA

2641 ATAGATTTAA ATGAAAATAC AGGTATTTGG GTCGTATTTA AAATTCCGAC AACAAATGGA

2701 TACGCAACAT TAGGAAACCT TGAATTGGTA GAAGAGGGGC CATTATCAGG AGACGCACTA

2761 GAACGCTTGC AAAGAGAAGA ACAGCAGTGG AAGCTTCAAA GAACCAAAAG ACGTGAAGAG

2821 ACGGATAGAA AATATATGGC AGCAAAACAA GCCATTGATC GTTTATTCGC AGATTATCAA

2881 GACCAACAAC TCAATTCTGG TGTAGAAATG TCAGATTTGC TTGCAGCTCA AAACCTTGTA

2941 CAGTCCATTC CTTATGTGTA TAACGAAATG TTCCCAGAAA TCCCTGGAAT GAACTATACA

3001 AATTTCACAG AGTTAACAAA CAGACTCCAA CAAGCATGGA ATTTGTATGA TCTTCGAAAT

3061 GCTATACCAA ATGGAGATTT TCGAAATGGA TTAAGTGATT GGAATGCAAC ATCAGATATA

3121 AATGTGCAAC AACTAAACGA TACATCTGTC CTTGTCATTC CAAACTGGAA TTCTCAAGTG

3181 TCACAACAAT TTACAGTTCA ACCGAATTAT AGATATGTAT TACGTGTCAC AGCGAGAAAA

3241 GAGGGAGCAG GAGACGGATA TGTGATCATC CGTGATGGTA CAAATCAGAC AGAAACACTC

3301 GCATTTAATA CATGTGATAA TGATGCAGGT GTTTTATCTA CTAATCAAGC TAGCTATATC

3361 ACAAAAACAG TGGAATTCAC GCCATCTACA GAGCAAGTTT GGATTGACAT GAGTGAGACC

3421 GAAGGTGTAT TCAACATAGA AAGTGTAGAA CTCGTGTTAG AAGAAGAGTA AG

                                                            

SEQ ID N02 (amino acid sequence of Cry8Ga1 protein):

1 MSPNNQNEYE IIDASSPTSV SNNSVKYPLA SDQTTTLQNM NYKDYLRMSE GENPELFGNP

61 ETFISAQDAV GTGIDIVSKL LGSLGVPLVG QAATALKWI I GKLWPSSGNP WDDLMTAVEE

121 LINQKIEAYA RSKALAELGV SGRAVKSYQT ALEEWQKNPN NARSAALVRE RFSDAEHTLR

181 TQMSLFTVRG YEIPLLATYA QAANLHLFVM KDIQIYGREW GYTQGDINLF YREQVEFTGE

241 YSDYCVKWYN AGLDKLRGST ALQWINYNRF RREMTVMALD IVALFPNYDI RMYPMKTTAE

301 LTRRIYTDPL GYTGSGSNTP PWYNYGYSFS WIENNAVPAP GLFQWLQGIG IYTKFARITP

361 FYANYWSGHT VFYKFTNDST ERRVQYGDTD TPELDSSSFE NVDIYKVSAS VGSYKSNTVL

421 LPTFKATFEG VNQNNQLKTF RYQKESNVPS QTKNSTTELP VQLSTPPTYG DSEQYSHRLA

481 YVFDAPIDSY TGIYRMYGFA PILGWTHISV SRDNRIDPDK ITQIPAVKAY AEGLANYIKD

541 PGFTGGDLLA LGRNSNTSLI VNFSKPQTYR IRIRYAASKT SYFQLRGLHN IAQSQRFEAT

601 YSNKNENDLT FNDFKYVEIQ KTVSIDNPSE SRSISIYTQS DTEYLLWTNR IHPSRCNIWS

661 GTRPRCGKES GEWLVYQYKR CLTDKCNGLS SQSSGKLSRM PIDELYPNEK RMLWDAVKEA

721 KRLVQARNLL QDTGFNVING ENGWTGSTGI EVVEGDVLFK DRSRLPSAR EIDTETYPTY

781 LYQQIDESLL KPYTRYRLRG FIGSSQDLEI KLIRHRANQI VKNVPDNLLP DVRPVNSCGG

841 VDRCSEQQYV DANLALENNG ENRNMSSDSH AFSFHMDTGE IDLNENTGIW VVFKIPTTNG

901 YATLGNLEV EEGPLSGDAL ERLQREEQQW KLQRTKRREE TDRKYMAAKQ AIDRLFADYQ

961 DQQLNSGVEM SDLLAAQNLV QSIPYVYNEM FPEIPGMNYT NFTELTNRLQ QAWNLYDLRN

1021 AIPNGDFRNG LSDWNATSDI NVQQLNDTSV LVIPNWNSQV SQQFTVQPNY RYVLRVTARK

1081 EGAGDGYVII RDGTNQTETL AFNTCDNDAG VLSTNQASYI TKTVEFTPST EQVWIDMSET

1141 EGVFNIESVE LVLEEE*

SEQ ID N03 (the nucleotide sequence of the artificially designed gene)

BamHI

1 CGC GGATCC GCGATGAGTCCGAATAACCAGAACGAATATGAGATCATCGATGCTTCCTCC

61 CCCACCTCTGTTTCTAATAACTCAGTGAAGTACCCCTTGCAAGCGACCAGACGACCACA

121 TTGCAAAACATGAACTACAAAAGATTACCTGCGGATGTCTGAGGGAGAGAATCCTGAACTT

181 TTTGGAAATCCCGAGACGTTCATTAGTGCTCAGGACGCTGTTGGCACTGGTATTGATATT

241 GTGAGCAAGCTGTTGGGTAGTTTGGGGGTTCCACTTGTTGGTCAGGCCGCCACGGCACTT

301 AAATGGATCATAGGTAAGTTGTGGCCTTCTTCCGGAAACCCCTGGGACGATTTGATGACG

361 GCCGTAGAAGAACTCATCAACCAGAAGATAGAAGCGTATGCCAGAAGCAAGGCACTTGCT

421 GAATTGGGTGTTAGCGGACGGGCTGTGAAGTCCTACCAAACCGCCCTTGAAGAGTGGCAG

481 AAGAACCCCAATAACGCTCGAAGCGCAGCCCTTGTCAGGGAAAGATTTTCAGACGCAGAA

541 CACACATTGCGTACCCCAAATGAGTTTGTTCACCGTTCGTGGTTACGAAATTCCGCTTTTA

601 GCCACATATGCACAAGCTGCCAATTTGCACTTGTTTGTGATGAAGGATATTCAAAATTTAC

661 GGTAGAGAATGGGGATACACTCAGGGCGACATCAACCTTTTTCTACAGGGAACAAGTAGAA

721 TTCACAGGGGAATACTCTGATTATTGCGTTAAGTGGTACAACGCTGGCTTGGACAAACTT

781 AGAGGCAGCACCGCTCTACAATGGATTAACTACAATAGGTTCCGCAGAGAAATGACAGTG

841 ATGGCCCTGGATATCGTTGCACTTTTTCCCCAATTACGACATCCGCATGTATCCAATGAAG

901 ACAACCGCAGAACTTACGCGAAGAATTTACACAGACCCCCTTGGTTACACGGGAAGCGGT

961 TCTAACACGCCACCATGGTATAACTACGGATACTCTTTCTCCTGGATAGAAAACAACGCC

1021 GTGCCAGCCCCTGGATTGTTCCAGTGGTTGCAAGGCATTGGGATCTATACCAAGTTTGCT

1081 CGTATCACTCCATTTTACGCGAACTACTGGTCAGGACATACTGTCTTTCTACAAATTTACT

1141 AACGATTCTACTGAGAGACGTGTTCAGTATGGAGACACAGATACTCCAGAATTAGACAGT

1201 TCTTCCTTCGAAAACGTTGACATTTATAAGGTTTCCGCCTCAGTTGGTTCGTACAAGAGGC

1261 AATACCGTGTTGTTACCAACTTTTAAAGCTACTTTCGAGGGTGTCAATCAGAACAATCAG

1321 CTTAAGACCTTTAGGTATCAAAAGGAATCTAATGTCCCAAAGCCAAACGAAGAACTCAACC

1381 ACAGAGCTGCCTGTTCAGTTGTCCACTCCGCCTACTTACGGAGACTCTGAACAGTACAGT

1441 CACAGACTAGCCTACGTTTTCGATGCCCCAATCGACTCATATACAGGCATATACCGCATG

1501 TACGGCTTTGCCCCTATTCTTGGTTGGACACACATTAGCGTGAGTAGGGACAACAGGATC

1561 GATCCAGACAAGATTACTCAAATTCCAGCTGTAAAGGCATATGCTGAGGGTCTTGCTAAT

1621 TACATCAAAGATCCCGGGTTCACAGGAGGCGACTTACTTGCTTTAGGTAGAAACTCCAAT

1681 ACTTCATTGATTGTCAACTTTTCGAAGCCTCAAACATACCGTATCCGTATTAGGTACGCT

1741 GCTAGCAAAACTTCGTATTTCCAACTACGTGGTCTGCACAATATCGCTCAGTCCAGCGT

1801 TTCGAAGCTACGTACTCTAATAAGAACGAAAACGATTTGACATTTAACGACTTCAAGTAC

1861 GTCGAAATTCAAAAAACTGTTTCCATCGACAATCCATCAGAAAGCAGGAGTATATCCATC

1921 TACACTCAATCAGATACAGAACTTCTTATTATAGACAAAAATCGAATTCATCCCAGTAGAC

1981 GCCACATTTGAAGCGGAACAAGACCTAGATGTGGCAAAGAAGGCTGTGAATGGCTTGTTC

KpnI SacI

2041 ACGAACACAAAGTAACGG GGTACC CCGA GCTCG

SEQ ID N04 (amino acid sequence of artificially designed gene mCry8Ga1 protein)

1 MSPNNQNEYE IIDASSPTSV SNNSVKYPLA SDQTTTLQNM NYKDYLRMSE GENPELFGNP

61 ETFISAQDAV GTGIDIVSKL LGSLGVPLVG QAATALKWII GKLWPSSGNP WDDLMTAVEE

121 LINQKIEAYA RSKALAELGV SGRAVKSYQT ALEEWQKNPN NARSAALVRE RFSDAEHTLR

181 TQMSLFTVRG YEIPLLATYA QAANLHLFVM KDIQIYGREW GYTQGDINLF YREQVEFTGE

241 YSDYCVKWYN AGLDKLRGST ALQWINYNRF RREMTVMALD IVALFPNYDI RMYPMKTTAE

301 LTRRIYTDPL GYTGSGSNTP PWYNYGYSFS WIENNAVPAP GLFQWLQGIG IYTKFARITP

361 FYANYWSGHT VFYKFTNDST ERRVQYGDTD TPELDSSSFE NVDIYKVSAS VGSYKSNTVL

421 LPTFKATFEG VNQNNQLKTF RYQKESNVPS QTKNSTTELP VQLSTPPTYG DSEQYSHRLA

481 YVFDAPIDSY TGIYRMYGFA PILGWTHISV SRDNRIDPDK ITQIPAVKAY AEGLANYIKD

541 PGFTGGDLLA LGRNSNTSLI VNFSKPQTYR IRIRYAASKT SYFQLRGLHN IAQSQRFEAT

601 YSNKNENDLT FNDFKYVEIQ KTVSIDNPSE SRSISIYTQS DTEYLLWTNR IHPSRCNIWS

661 GTRPRCGKES GEWLVYQYKR

Claims (12)

1. Bacillus thuringiensis strain HBF-18, its preservation number is: CGMCC2070. 2. The nucleotide sequence of the Bacillus thuringiensis cry8Gal gene highly effective against coleopteran pests is shown in SEQ ID NO.1. 3. An engineering bacteria strain BioT8G, characterized in that it contains the cry8Gal gene according to claim 2. 4. The Bacillus thuringiensis cry8Gal protein highly effective for coleopteran pests is encoded by the cry8Gal gene of claim 2, and its amino acid sequence is as shown in SEQ ID NO.2. 5. The application of the cry8Gal protein according to claim 4 in the preparation of a medicament for killing coleopteran pests. 6. a kind of albumen, has the identical function of albumen described in claim 4, and its aminoacid sequence is as shown in SEQ ID NO4. 7. a kind of artificial transformation synthetic mcry8Gal gene, the protein described in its coding claim 6, its nucleotide sequence is as shown in SEQ ID NO.3. 8. A plant expression vector pBSmGN is characterized in that this plant expression vector consists of the mcry8Gal gene sequence described in claim 7, the constitutive expression promoter CaMV35S, the NOS terminator and a kind of gene that can be used in Escherichia coli and Agrobacterium tumefaciens The shuttle binary vector pBI121 was constructed. 9. The application of the mcry8Gal gene according to claim 7 in plant resistance to coleopteran pests. 10. The application according to claim 9, characterized in that the plant expression vector pBSmGN according to claim 8 is transformed into plants, so that the plants can produce toxicity against coleopteran pests. 11. Use according to claim 10, said plant being tobacco. 12. The application according to claim 9, characterized in that the protein expressed by the mcry8Gal gene is prepared into a medicament for killing coleopteran pests.

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