CN103266069B - Bacillus thuringiensis strain and application thereof - Google Patents

Abstract

The invention provides a Bacillus thuringiensis strain TD516, the preservation number of which is CGMCC No.2859. The strain has activity against Diptera insects. The invention also provides the application of the strain TD516 for preparing insecticides. The insecticides containing TD516 can reduce the usage of chemical insecticides and reduce environmental pollution, and have important economic value and application prospect.

Description

A Strain of Bacillus thuringiensis and Its Application

This application is a divisional application of a patent application with an application date of November 29, 2011, an application number of 201110387753.9, and an invention titled "A Bt protein Cyt3Aa1, its encoding gene and its application".

technical field

The invention relates to the field of biotechnology, in particular to a novel Bt protein and its coding gene and application.

Background technique

For many years, humans have generally used chemical control methods to prevent and control mosquitoes. However, due to the long-term and large-scale use of chemical pesticides, they have polluted the environment and brought harm to human survival and health. In addition, while chemical pesticides kill pests, they also kill natural enemies and other beneficials, destroying the ecological balance. Compared with chemical control, biological control is safe, effective and durable, and avoids a series of problems caused by chemical control. Among biopesticides, Bacillus thuringiensis is currently the most widely used and most productive type of microbial pesticide in the world.

Bacillus thuringiensis (Bt for short) is a Gram-positive bacterium with a wide distribution. It can form parasporal crystals composed of proteins with insecticidal activity during the formation of spores, also known as insecticidal crystals. Proteins (Insectididalcrystal proteins, ICPs for short) are encoded by the cry gene and the cyt gene. More than 470 genes encoding insecticidal crystal proteins have been isolated and cloned, and they are respectively identified as different groups, subgroups, classes and subclasses according to the homology of the encoded amino acid sequences (Crickmore N., et al.1998. Microbiol Mol Biol Rev, 62:807-813; http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/). Cyt protein was first discovered in Bt Israel subspecies, which is toxic to Diptera insects, and it is composed of delta-endotoxin produced by Bt in the spore stage. As of December 2010, a total of 31 Cyt proteins have been discovered, which can be divided into two categories, Cyt1 and Cyt2, according to the amino acid sequence homology. The measurement of Cyt protein's insecticidal activity against Aedes aegypti shows that: Cyt1Aa, Cyt1Ab1, Cyt2Aa1 and Cyt2Ba1 are all active against Aedes, but the activity of Cyt1 protein is higher than that of Cyt2 (Qi Donglai, Li Yidan, Gao Jiguo.2005.Journal ofNortheast Agricultural University. 12(1):5-10.). Cyanobacteria, which can be used as food for mosquito larvae, are widely grown in natural water bodies. In 1996, Yan Ge and others introduced the pDC26 shuttle plasmid carrying the Bt insecticidal crystal protein gene into a variety of Anabaena for expression by conjugative transfer. 7120, Anabaena cylidrica ^and Anabaena subtropic, the 24 ^- h median lethal concentrations (LC ₅₀ ) of the obtained genetically engineered algae Anabaena sp. 4.34×10 ⁴ cells/mL. In the case of more than 50 continuous passages, the engineered algae can still have a high poisonous effect on the larvae of Culex palustris; in the absence of antibiotics, the shuttle plasmid is stable, and the level of toxic effect on mosquito larvae is comparable to that of engineering cultured with antibiotics. There is little difference between algae and algae, which provides a new way for the study of continuous control of mosquito larvae.

Because Cyt protein and Cry protein have different action principles, Cyt protein has a synergistic effect on some Cry proteins, which has attracted more and more attention. Studies have found that the effect of Cyt protein on Diptera pests is not mainly in its virulence itself, but in that it can enhance the insecticidal activity of other insecticidal crystal proteins, for example: the relationship between Cyt1Aa protein and other Cry proteins of Bt Israel subspecies There is an obvious synergistic effect. Cyt1Aa is mixed with other insecticidal proteins of Bt Israel subspecies in different proportions, and the toxicity of a single protein is found to be 4 to 10 times higher (Crickmore N, et al.1995. FEMS Microbiol Lett, 131:249-254).

Bacillus thuringiensis has been widely used in the control of crops and horticultural plant pests, forest pests and sanitary pests, and also has good results. However, the inevitable result of frequently using a single Bt insecticidal protein is the resistance to Cry proteins due to the adaptation of pests; at present, many insect populations have successively developed resistance to insecticidal crystal proteins to varying degrees. Since the mid-1980s, the problem of resistance has been confirmed in laboratory and field experiments (Mc Gaughey W.H., 1985. Science. 229:193-195), mainly due to the continuous use of single species and sublethal doses of Bt And the application of Bt transgenic insect-resistant plants has caused insect populations to be subject to the selection pressure of insecticides for a long time. Since the 1990s, it has been confirmed for the first time in Hawaii that the diamondback moth in the field has developed significant resistance to Bt insecticides (Tabashnik B.E., et al.1994.Proc.Natl.Acad.Sci.USA.91:4120-4124 ); in Shenzhen, Guangzhou, Shanghai and other places where Bt insecticides have been used for a long time in my country, it was found that the control effect of Bt insecticides on Plutella xylostella significantly decreased, which means that resistance has been formed (Feng Xia. 1996. Guangdong Plutella xylostella against Resistance of Bacillus thuringiensis. Acta Entomological Sinica, 39(3):238-244). At present, it is found that at least a dozen kinds of insects have developed resistance to Bt and its insecticidal crystal protein in the laboratory and in the field. The mathematical model of selection pressure is used to predict that under the condition of selection pressure of Bt transgenic insect-resistant plants, insects will Produce resistance (Schnepf E., et al. 1998. Microbiol. Mol. Biol. Rev. 65 (3): 775-806). Studies have shown that Cyt protein can reduce the resistance of Diptera pests to Cry protein: the mixture of Cyt1A and Cry11A can reduce the resistance of Culexquinquefasciatus (Culexquinquefasciatus) resistant line to Cry11A by 1000 times, while Cyt1A and Cry4, Cry11A Mixed use can completely inhibit the resistance of Cry4 and Cry11A of the Cry4 and Cry11A resistant lines of Culex pentapatina. After 28 generations of Culex pentapipes selected by the crystal protein (containing Cyt1Aa protein) of subspecies Israel, the resistance was only increased by 3 times; but if the crystal protein without Cyt1Aa was used, the resistance would be increased by 90-900 times. Since the Israeli subspecies has been used to kill mosquitoes, no obvious resistance has been found so far, and the Cyt1Aa protein plays a key role.

It is precisely because of the unique anti-mosquito larvae effect of Cyt protein, as well as its ability to enhance the virulence of Cry protein and reduce or delay Cry protein resistance, it will play an important role in the long-term management of Cry protein resistance, looking for new cyt proteins Gene resources are of great significance to my country's biological control.

Contents of the invention

The first object of the present invention is to provide a new Bt virulence protein Cyt3Aa1.

The second object of the present invention is to provide a gene encoding the protein.

The third object of the present invention is to provide the application of the above protein and gene.

The present invention is a new bacterial strain TD516 of Bacillus thuringiensis isolated from soil in Muchuan primeval forest area of Sichuan Province. The strain was preserved on January 12, 2009 at the General Microorganism Center of China Committee for Culture Collection of Microbial Cultures (CGMCC for short, address: Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101), and the classification was named Su Yunjin Bacillus (Bacillusthuringiensis), the preservation number is CGMCC No.2859.

The toxicity test of TD516 shows that TD516 has extremely high toxicity to Diptera pests. Design a pair of specific primers according to the conserved sequence of the cyt3 gene, and amplify its genomic DNA. The results show that the strain has the cyt3 gene. Further design its full-length gene primers and clone the cyt3Aa1 gene. Its nucleotide sequence is shown in the sequence table SEQ ID As shown in No.1, the full length is 777bp, and the analysis shows that the GC content is 32.56%, encoding a protein consisting of 258 amino acids. After determination, its amino acid sequence is shown in SEQ ID No.2. It was officially named cyt3Aa1 by the International Bt Insecticidal Crystal Protein Gene Nomenclature Committee. The amino acid composition of Cyt3Aa1 protein is shown in Table 1. The active center of the protein is located in the (2) segment.

Amino acid composition of table 1 Cyt3Aa1 protein

amino acid percentage/% amino acid percentage/% Ala(A): 3.88 Met(M): 0.78 Cys(C): 1.94 Asn(N): 7.36 Asp(D): 3.88 Pro(P): 5.81 Glu(E): 2.71 Gln(Q): 4.26 Phe(F): 6.89 Arg(R): 4.26 Gly(G): 5.04 Ser(S): 6.98 His(H): 2.71 Thr(T): 6.59 Ile(I): 6.20 Val(V): 6.20 Lys(K): 8.53 Trp(W): 0.39 Leu(L): 10.47 Tyr(Y): 5.04

It should be understood that those skilled in the art can substitute, delete and/or add one or several amino acids according to the amino acid sequence (SEQ ID No.2) of the protein Cyt3Aa1 disclosed in the present invention without affecting its activity to obtain the The mutant sequence of the protein. For example, in the inactive segment, replacing Asn at position 16 with Ala, replacing Pro at position 25 with Ser, deleting Val at position 49, adding a Gln or adding Ala at position 60 will not affect its activity . Therefore, the Bt protein Cyt3Aa1 of the present invention also includes a protein derived from Cyt3Aa1 having the same activity as the Bt protein Cyt3Aa1 after the amino acid sequence shown in SEQ ID No. 2 is substituted, substituted and/or increased by one or several amino acids.

The gene of the present invention includes the nucleotide sequence encoding the protein Cyt3Aa1.

The present invention provides the gene encoding the above-mentioned Bt protein Cyt3Aa1, the nucleotide sequence of which is shown in the sequence table SEQ ID NO.1.

In addition, it should be understood that, considering the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for the expression of specific species as needed.

The gene and protein of the present invention can be cloned or isolated from strain TD516, or obtained by DNA or peptide synthesis.

The gene of the present invention can be operably connected with the expression vector to obtain a recombinant expression vector capable of expressing the protein of the present invention, and then the expression vector can be introduced into the host through transgenic methods such as click method and gene gun method to obtain the cyt3Aa1 gene-transferred Transformants, such as aquatic organisms such as Anabaena, confer insect resistance activity.

In one embodiment of the present invention, the Bt protein Cyt3Aa1 recombinant expression vector is obtained by inserting the cyt3Aa1 gene into the expression vector pET-32a(+) to construct the recombinant expression vector pET-3Aa.

In addition, the fermentation broth containing Cyt3Aa1 protein can also be obtained by fermenting the bacterial strain TD516 of the present invention, which can be prepared into an insecticide for the control of crop pests. Those skilled in the art can also transform the above-mentioned gene into bacteria or fungi, and produce the Bt protein of the present invention through large-scale fermentation. Therefore, the present invention provides the application of the Bt protein Cyt3Aa1 or its coding gene cyt3Aa1 or the recombinant expression vector containing the gene in the preparation of pesticides.

The invention provides the application of the Bt protein Cyt3Aa1 or its coding gene cyt3Aa1 or the recombinant expression vector containing the gene in the preparation of transgenic aquatic organisms (such as Anabaena).

Those skilled in the art can also transform aquatic organisms (such as Anabaena) according to the cyt3Aa1 gene disclosed in the present invention, so that they have corresponding insect-resistant activities.

The invention provides that the Cyt3Aa1 protein is a new Bt protein, which has good insecticidal activity. It is used to prepare transgenic aquatic plants, which can specifically kill pests, reduce the amount of pesticides used, reduce costs, and reduce environmental pollution. . At present, there is no report that pests or insects develop resistance to this protein. Therefore, the Bt protein Cyt3Aa1 of the present invention has important economic value and application prospect, and is suitable for large-scale application in controlling mosquito larvae.

Description of drawings

Figure 1 is the electrophoresis diagram of the PCR amplification result of the cyt3Aa1 gene, wherein M.DNAmarker, 1.cyt3Aa1 gene;

Figure 2 is the enzyme digestion identification diagram of recombinant plasmid PET-3Aa, 1. Recombinant plasmid pET-3Aa, 2. Kpn I+Sal I double enzyme digestion pET-32a, 3. Kpn I+Sal I double enzyme digestion pET-3Aa, 4. Inserted DNA, M.DNA marker;

Figure 3 is the SDS-PAGE detection of cyt3Aa1 gene in E.coli BL21(DE3), in which M. protein marker; 1. Expression of empty vector (pET-32a) in E.coii BL21(DE3), 2. Lysis 3. Cyt3Aa1 protein in inclusion bodies.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

Cloning of embodiment 1 cyt3Aa1 gene

The new bacterial strain TD516 of Bacillus thuringiensis isolated from the soil in the virgin forest area of Muchuan, Sichuan Province in the present invention has been collected in the General Microorganism Center of China Microbiological Culture Collection Management Committee (Address: Chaoyang District, Beijing) on January 12, 2009. Datun Road, Institute of Microbiology, Chinese Academy of Sciences, Zip code 100101) is preserved, the classification is named Bacillus thuringiensis (Bacillus thuringiensis), and the preservation number is CGMCC No.2859.

The full-length sequence of the cyt3Aa1 gene was obtained by cloning as follows.

The total DNA of strain TD516 was extracted using a genomic DNA purification kit (purchased from Saibaisheng Company). And design the primer sequence as follows:

P1: 5'ATGTATACTAAAAATTTTAGTAAG3'

P2: 5'TTACGAAAACTTTAAAATTATGAAT3'

25μl PCR reaction system:

10×buffer 2.5μl

MgCl ₂ (25mM) 1.5μl

Taq enzyme 0.2μl

dNTPs (2.5mM) 2μl

Upstream primer P1 1μl

Downstream primer P2 1μl

Template 5 μl

Double distilled water 11.8μl

Thermal cycle reaction: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 1 minute, annealing at 45°C for 50 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 10 minutes; stop reaction at 4°C. The amplification reaction products were electrophoresed on 1% agarose gel, and the PCR amplification results were observed in a gel imaging system. The results are shown in Figure 1. A sequence of about 800 bp was obtained through amplification, and the sequence was sequenced. The nucleotide sequence is shown in SEQ ID No.1, which is consistent with the size of the target sequence.

Example 2 Obtaining of Cyt3Aa1 protein

According to the two ends of the open reading frame of Cyt3Aa1 gene, a pair of specific primers were designed and synthesized: cry3F: 5'-CGG GGTACC ATGTATACTAAAAATTTTAAGTAAG-3cry3R: 5'-CGC GTCGAC TTACGAAAACTTTAAAATTATGAAT-3', the bases underlined at the 5' end primers are respectively Kpn I and Sal I restriction sites. Using TD516 plasmid DNA as a template for amplification, the amplified product was double-digested with Kpn I and Sal I, and the digested product was ligated with the vector pET-32a(+) after the same double-digestion, and transformed into E.coli DH5α For competent cells, the recombinant plasmid was named pET-3Aa. Recombinant plasmids were digested and electrophoresis verified that the size of the inserted fragments met the target fragments (Figure 2) before being transferred into the recipient strain E.coli.BL21(DE3), and the recombinants containing the recombinant plasmids were named E.coli.BL21(3Aa). Then the positive transformant was transferred to LB medium, under the culture condition of 210r/min, 37°C, when the OD ₆₀₀ value was 0.6, 0.6mmol/L IPTG was added to induce expression for 20h. SDS-PAGE analysis showed that the expression product of cyt3Aa1 gene had a molecular weight of about 30kDa in the precipitate after sonication of the bacteria (Figure 3), which was consistent with the predicted protein molecular weight.

Anti-insect effect of embodiment 3Cyt3Aa1 protein

The Cyt3Aa1 protein obtained in Example 2 was used to measure the insecticidal activity against cotton bollworm, black beetle, North China black beetle and Aedes mosquito respectively.

For Lepidoptera pests: the Cyt3Aa1 protein obtained in Example 2 was formulated into 6 different concentrations from 1 μg/ml to 100ng/ml, and the old and tender cabbage leaves were selected to be washed and dried; Cut into 2×2cm ² size, soak in different concentrations of Cyt3Aa1 protein solution for 5 minutes; take out and drain the excess liquid, put it in a sterilized petri dish to dry, use the leaves soaked in LB liquid medium as a control, each Put 4 leaves in a petri dish; select 30 healthy 2-3 instar cotton bollworms in each petri dish; repeat each treatment 3 times, put them indoors, observe the larval death after 3 days, and calculate LC ₅₀ with SPSS10.0 software.

For Coleopteran pests: the Cyt3Aa1 protein obtained in Example 2 was formulated into 6 different concentration gradients of 0.1, 4, 8, 16, 32, and 64 μg/mL. Then add the diluted solution to the sterilized fine soil of potato shreds of uniform thickness and mix evenly. With 5-7 days old black beetle and North China large black beetle as the main insects for testing, 30 worms are placed in each treatment group, and repeated Three times, the treatment of adding water was used as the blank control, and the number of dead insects was checked after 7 days and 14 days of feeding, and the LC ₅₀ was calculated by SPSS10.0 software.

For Diptera pests: the Cyt3Aa1 protein obtained in Example 2 was prepared into 6 different concentration gradients of 0.1, 4, 8, 16, 32, and 64 μg/mL, and then 20 Aedes larvae were put into each treatment, and each The treatment was repeated three times, and clear water was used as the blank control; after 12 hours, the death of Aedes larvae was counted, and the LC ₅₀ was analyzed with SPSS10.0 software.

The results showed that Cyt3Aa1 protein had almost no insecticidal activity against cotton bollworm and black beetle, but had the best killing activity against Aedes larvae, with an LC ₅₀ of 3.25 μg/mL.

Claims (8)

1. Bacillus thuringiensis (Bacillus thuringiensis) strain TD516, its preservation number is CGMCC No.2859, this bacterial strain contains protein Cyt3Aa1, and its amino acid sequence is shown in SEQ ID NO.2. 2. the microbial agent that contains bacillus thuringiensis TD516 described in claim 1. 3. The application of Bacillus thuringiensis TD516 or its fermentation product in the preparation of insecticides according to claim 1, wherein the insects are Diptera pests. 4. The application of the microbial agent according to claim 2 in the preparation of insecticides, wherein the insects are Diptera pests. 5. The application of Bacillus thuringiensis TD516 or its fermentation product in claim 1 in improving insect resistance of aquatic organisms, wherein the insects are Diptera pests. 6. The application of the microbial agent as claimed in claim 2 in improving the insect resistance of aquatic organisms, wherein the insects are Diptera pests. 7. The application according to claim 5 or 6, wherein the aquatic organisms are phytoplankton, aquatic algae or aquatic microorganisms. 8. An insecticide having a killing effect against dipteran pests, characterized in that it contains Bacillus thuringiensis TD516 according to claim 1.