CN105367634B - A kind of Bt PROTEIN C ry1Ie5, its encoding gene and application - Google Patents

Abstract

The present invention provides a Bt protein Cry1Ie5 and its coding gene, said protein has the amino acid sequence shown in SEQ ID No.2, or the amino acid sequence shown in SEQ ID No.2 is substituted, deleted and/or added with one or Amino acid sequences with multiple amino acids and equivalent activity. The protein of the present invention can be used to prepare Bt insecticide, and the gene encoding the protein can transform crops such as cotton, corn, rice, vegetables, etc., so that it has corresponding insect-resistant activity, thereby reducing the use of pesticides, reducing environmental pollution, and having Important economic value and application prospect.

Description

A kind of Bt protein Cry1Ie5, its coding gene and application

technical field

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

Background technique

In the process of human production, insect pests are an important factor that causes agricultural production losses and affects human health. According to the statistics of FAO, the annual economic loss caused by insect pests in the world's agricultural production is as high as 14%, the loss of diseases is 12%, and the loss of weeds is 11%. The loss was as high as 126 billion US dollars, equivalent to half of China's total agricultural output value, and more than four times that of the United Kingdom. In order to reduce these losses, for many years, chemical control methods have been widely used to control crop pests and mosquitoes. However, due to the long-term and large-scale use of chemical pesticides, the pollution to the environment has been caused, and the amount of pesticide residues in agricultural by-products has increased. and health hazards. 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 avoid a series of problems caused by chemical control. Therefore, biological control technology has become a research hotspot. 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 very wide distribution. When spores are formed, they can form parasporal crystals composed of proteins with insecticidal activity, also known as insecticidal crystals. Proteins (Insectididal crystal proteins, ICPs for short), ICPs are encoded by the cry gene, are highly toxic to sensitive insects, but non-toxic to higher animals and humans. At present, Bt has become a powerful substitute for chemically synthesized pesticides in the control of farmland pests, forest pests and sanitary pests. Bt is also an important gene source for transgenic insect-resistant engineering plants.

Since Schnepf cloned the first gene capable of expressing insecticidal activity from the strain HD-1 in 1981, more than 500 genes encoding insecticidal crystal proteins have been isolated and cloned, and they are classified according to the homology of the encoded amino acid sequence. identified as distinct groups, subgroups, classes and subclasses, respectively (Crickmore N, et al. Microbiol Mol Biol Rev, 1998, 62:807-813; http://www.biols.susx.ac.uk/Home/ Neil_Crickmore/Bt/). Generally speaking, toxic proteins such as Cry1, Cry2 and Cry9 are effective against Lepidoptera pests; among them, Cry1 and Cry9 proteins are the most studied, and the insecticidal crystal proteins encoded by them have a molecular weight of 130-140kD, and many genes have been widely used. Control of Lepidoptera pests applied to plants. The toxin protein produced by Bacillus thuringiensis subsp. israelensis (Bti for short) has good insecticidal activity against mosquitoes and is widely used in mosquito control. At the same time, the Cyt protein is cytolytic, has a synergistic effect on some Cry proteins and delays insect resistance.

Insecticides based on Bt insecticidal crystal protein have been used for more than 50 years. At first, insect resistance to Bt was not detected. Confirmed in field experiments (McGaughey, WH 1985. Science. 229:193-195), the main reason is that the continuous use of single species and sublethal doses of Bt and the application of Bt transgenic insect-resistant plants have caused long-term exposure of insect populations to insecticides. selection pressure. In 1985, McGaughey reported that under the selection pressure of Dipel (commercial preparation of Btsubsp. kurstaik HD-1), the warehouse grain pest Plodia interpunctella , the resistance increased 97 times after 15 generations; under high dose selection pressure , the resistance can be increased by 250 times. In 1990, it was first confirmed in Hawaii that the diamondback moth in the field had developed significant resistance to Bt insecticides (Tabashnik, BE, et al. 1994. Proc.Natl.Acad.Sci.USA.91:4120-4124), Since the 1990s, in Shenzhen, Guangzhou, Shanghai and other places where Bt insecticides have been used for a long time in China, it has been found that the control effect of Bt insecticides on diamondback moth has decreased significantly, which means that resistance has formed (Feng Xia. 1996. Acta Entomology, 39 (3):238-244; Hofte, H., 1988. Appl. Environ. Microbiol. 54: 2010-2017). 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. It is predicted by the mathematical model of selection pressure that under the condition of selection pressure of Bt transgenic insect-resistant plants, insects will produce Resistance (Schnepf, E., et al. 1998. Mol. Biol. Rev.65 (3):77 5-806). In addition, studies have shown that resistance to Bti has not been found in field use, but mosquito resistance to it has been continuously confirmed in the laboratory, and this situation may also appear in field (Georghiou GP, 1997. Applied and Environmental Microbiology, 63:1095-1101.).

In order to avoid losses caused by resistant insects, finding new highly virulent Bt gene resources is an effective way to solve this problem, which is of great significance to the biological control of our country.

Contents of the invention

The first object of the present invention is to provide a new Bt virulence protein resource to address the above-mentioned deficiencies.

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 new strain BN23-5 of Bacillus thuringiensis isolated from the soil in Chengdu City, Sichuan Province in the present invention has been collected in the General Microorganism Center of China Committee for Microorganism Culture Collection (CGMCC for short, address: Beijing) on July 14, 2014. No. 3, No. 1 Yard, Beichen West Road, Chaoyang District, City, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101) preserved, classified as Bacillus thuringiensis ( Bacillus thuringiensis ), and the preservation number is CGMCC No.9448.

The toxicity test of BN23-5 shows that BN23-5 has extremely high toxicity to Lepidoptera pests and the like. A pair of specific primers were designed according to the conserved sequence of the cry1I gene, and its genomic DNA was amplified. The results showed that the cry1I gene existed in the strain, and its full-length gene primers were further designed to clone the Cry1Ia-like gene. The nucleotide sequence is as follows: As shown in the list of SEQ ID No.1, the full length is 2160bp, and the analysis shows that the GC content is 36.81%, encoding a protein consisting of 720 amino acids. After determination, its amino acid sequence is shown as SEQ ID No.2. Prediction of the entire sequence using the bacterial sigma7.0promoter program on the softberry website showed that the sequence containing the activation site of RNA polymerase upstream of the coding region of the gene was named Cry1Ie5. The amino acid composition of Cry1Ie5 protein is shown in Table 1.

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 (SEQID No.2) of the protein Cry1Ie5 disclosed in the present invention without affecting its activity, to obtain the described The mutant sequence of the protein. For example, in the inactive segment, replacing Lys at position 44 with Arg, or replacing Leu at position 713 with Ile will not affect its activity. Therefore, the Bt protein Cry1Ie5 of the present invention also includes a protein derived from Cry1Ie5 that has the same activity as the Bt protein Cry1Ie5 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 Cry1Ie5 .

The present invention provides the gene encoding the above-mentioned Bt protein Cry1Ie5 , its nucleotide sequence is:

(1) the nucleotide sequence shown in SEQ ID NO.1 of the sequence listing, or

(2) The nucleotide sequence shown in SEQ ID No.1 is substituted, deleted and/or added by one or several nucleotides.

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 Bt bacterial strain BN23-5, 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 transformed into Introduce the host to obtain a transformant of the Cry1Ie5 gene, such as crops or fruit trees, so that it has insect-resistant activity.

In one embodiment of the present invention, the Bt protein Cry1Ie5 recombinant expression vector is obtained by inserting the Cry1Ie5 gene into the expression vector pET-28a(+) to construct the recombinant expression vector pET-1I.

In addition, the fermentation liquid containing Cry1Ie5 protein can also be obtained by fermenting the bacterial strain BN23-5 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.

The invention also provides the application of Bt protein Cry1Ie5 in improving plant insect resistance.

The invention provides the application of Bt protein Cry1Ie5 in cultivating transgenic plants.

Those skilled in the art can also transform crops such as cotton, corn, rice, and vegetables according to the Cry1Ie5 gene disclosed in the present invention, so that they have corresponding insect-resistant activities. For example: using the degeneracy of codons, the Cry1Ie5 gene is designed to have a gene sequence with rice preferred codons, and then the synthetic Cry1Ie5 gene sequence is connected to the vector pCAMBIA1300, and then transferred into the rice genome through Agrobacterium-mediated transformation, thereby obtaining Transgenic rice varieties with insect resistance activity.

The invention provides that the Cry1Ie5 protein is a Bt protein and has good insecticidal activity. It is used to prepare transgenic plants, which can specifically kill pests, reduce the use of pesticides, reduce costs, and reduce environmental pollution. During the effect verification test of the present invention, no pests were found to develop resistance to the protein. Therefore, the Bt protein Cry1Ie5 of the present invention has important economic value and application prospect, and is suitable for large-scale application in improving the insect resistance of plants.

Description of drawings

Figure 1 shows the gel electrophoresis image of the cloned Cry1Ie5 full-length gene, wherein M, DNA marker; 1, Cry1Ie5 gene;

Figure 2 shows the enzyme digestion identification map of the recombinant plasmid pET-1I, wherein 1, the BamH I+Hind III double digestion product of the recombinant plasmid pET-1I; 2, the vector pET-28a; 3, the recombinant plasmid pET-1I; 4, inserted DNA; M, DNAmarker;

Figure 3 shows the SDS-PAGE detection graph of Cry1Ie5 gene expression in E. coli BL21 (DE3); 1, the supernatant of the lysate of E. coli BL21 (DE3) containing the vector pET-28a induced by IPTG solution (negative control); 2, E.coli BL21(DE3) containing the vector pET-28a) was induced by IPTG and precipitated from the lysate (negative control); 3, E.coli BL21(DE3) containing the recombinant plasmid pET-1I ( DE3) supernatant of cell lysate induced by IPTG; 4, precipitation of cell lysate of E. coli BL21 (DE3) containing recombinant plasmid pET-1I induced by IPTG; M, protein marker.

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 biochemical reagents used in the examples are all commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

Cloning of Example 1 Cry1Ie5 Gene

The new strain BN23-5 of Bacillus thuringiensis isolated from the soil in Chengdu City, Sichuan Province according to the present invention, the strain has been collected on July 14, 2014 in the General Microorganism Center of China Microbiological Culture Collection Management Committee (Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Zip Code 100101) preserved, classified as Bacillus thuringiensis ( Bacillus thuringiensis ), and the preservation number is CGMCC No.9448.

In this example, the full-length sequence of the Cry1Ie5 gene was cloned by the following method.

The total DNA of strain BN23-5 was extracted using a genomic DNA purification kit (purchased from Saibaisheng Company) as a template for amplifying the Cry1Ie5 gene, and the primer sequences were designed as follows:

P1 (SEQ ID No. 3): 5'-ATGAAACTAAAGAATCAAGATAAG-3;

P2 (SEQ ID No.4): 5'-CTACATGTCACGCTCAATAT-3'

25μl PCR reaction system:

10 × buffer 2.5μl

MgCl ₂ (25mM) 1.5μl

Taq enzyme 0.2μl

dNTPs (2.5mM) 2μl

Primer P1 1μl

Primer P2 1μl

Template 5μl

Double distilled water 11.8μl

Thermal cycle reaction: pre-denaturation at 94°C for 5 minutes; 30 cycles of denaturation at 94°C for 50s, 50s at 54°C, and extension at 72°C for 2 minutes; 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 result is shown in FIG. 1 , a sequence of about 2160 bp was obtained through amplification, and the sequence was sequenced, and its nucleotide sequence was shown in SEQ ID No.1, which was consistent with the target sequence.

Example 2 Obtaining of Cry1Ie5 protein

According to the sequence of both ends of the open reading frame of Cry1Ie5 gene, a pair of specific primers 1ITF (SEQ ID No.5): 5'- GCC GGATCC ATGAAACTAAAGAATCAAGATAAG-3', 1ITR (SEQ ID No.6): 5'-CCCA AAGCTT CTACATGTCACGCTCAATAT was designed and synthesized The bases underlined at the -3' and 5' end primers are the restriction sites of BamH I and Hind III respectively. The total DNA of BN23-5 was used as a template to amplify, and the digested product was ligated with the vector pET-28a(+) after the same double digestion, and transformed into E. coli DH5α competent cells, and the plasmid was extracted and electrophoresed to verify the insertion After the size of the fragment meets the expected target fragment (Figure 2), it is then transferred into the recipient strain E.coli .BL21(DE3) (purchased from Beijing Quanshijin Biotechnology Co., Ltd.). The recombinant plasmid was named pET-1I, and the recombinant containing the recombinant plasmid was named E.coli .BL21(2L). Cultivate positive transformants in LB medium overnight at 200 r/min at 37°C, then transfer the culture solution to a 1L Erlenmeyer flask containing 400mL LB culture solution at a ratio of 1:100, at 200 r/min, Cultivate at 37°C, when the OD=600 value of the culture medium reaches 0.6-0.8, add 0.6 mmol/L IPTG to induce expression for 12 h, centrifuge the culture medium to collect the bacteria, discard the supernatant, add 30 mL 10 mmol/L Tris- HCl (pH 8.0) was sonicated, and the expressed protein was detected by SDS-PAGE.

SDS-PAGE analysis showed that the expression product of the gene was in the precipitate and supernatant after sonication (Figure 3), and the molecular weight of Cry1Ie5 was about 81.3 kDa, which was consistent with the predicted protein molecular weight.

Example 3 Cry1Ie5 protein insecticidal activity assay

The Cry1Ie5 protein obtained in Example 2 was tested for its insecticidal activity against diamondback moth and corn borer. Bioassay of Plutella xylostella: Cry1Ie5 protein was formulated into 6 different concentration gradients of 4, 2, 1, 0.5, 0.25, 0.01 ug/mL; selected old and tender cabbage leaves were washed and dried; irradiated under ultraviolet light 15 minutes, cut into 2×2cm ² size, put in different concentrations of bacteria solution, soak for 5 minutes; take out and drain the excess liquid, put it in a sterilized petri dish to dry, E.coli. BL21(DE3) as a negative control , clear water was the blank control, and 4 leaves were placed in each petri dish; 20 healthy 2-3 instar diamondback moths were selected and placed; each treatment was repeated 3 times, placed indoors, and the death of larvae was investigated after 3 days. Bioassay of corn borer: Cry1Ie5 protein was formulated into 6 different concentration gradients of 200, 100, 50, 25, 12.5, 0.1 ug/mL, and the protein was added to the feed for raising corn borer and mixed, E.coli. BL21 (DE3) As a negative control, clear water was used as a blank control, and then 20 2-3 instar corn borers were put into each treatment, and each treatment was repeated 3 times, and the results were counted after 7 days. LC50 was calculated with SPSS _10.0 software.

The results showed (Table 2): the expression product had a very high insecticidal activity against Plutella xylostella, with an LC ₅₀ of 0.43 ug/mL, and a high insecticidal activity against the corn borer, with an LC ₅₀ of 48.39 ug/mL; the bioassay results showed that , E.coli. BL21(DE3) and the blank control have no insecticidal activity against diamondback moth and corn borer.

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Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (8)

1. A Bt protein Cry1Ie5, the amino acid sequence of which is: the amino acid sequence shown in SEQ ID No.2. 2. A gene encoding the protein of claim 1. 3. The gene according to claim 2, which is as follows 1): 1) Its nucleotide sequence is shown as SEQ ID No.1 in the sequence listing. 4. The recombinant expression vector containing the gene of claim 2 or 3. 5. The insecticide containing the protein according to claim 1, wherein said insects are diamondback moth and corn borer. 6. the protein described in claim 1 or its coded gene is in the application of preparation insecticide, and described worm is side dish moths and corn borers. 7. Application of the protein or its coding gene according to claim 1 in cultivating transgenic plants. 8. the protein described in claim 1 or its coded gene is in the application in improving plant insect resistance, and described insect is diamondback moth and corn borer.