CN102603876B

CN102603876B - Bt protein Cry59Bal, coding gene and application of Bt protein Cry59Bal

Info

Publication number: CN102603876B
Application number: CN 201210017915
Authority: CN
Inventors: 郑爱萍; 李巧; 关鹏; 李平
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2012-01-19
Filing date: 2012-01-19
Publication date: 2013-08-07
Anticipated expiration: 2032-01-19
Also published as: CN102603876A

Abstract

本发明提供了一种新的Bt蛋白Cry59Ba1及其编码基因，所述蛋白具有SEQ ID No.2所示的氨基酸序列，或SEQ ID No.2所示的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸且具有同等活性的氨基酸序列。本发明蛋白可以用于制备Bt杀虫剂，编码该蛋白的基因可以转化棉花、玉米、水稻、蔬菜等农作物，使其具备相应的抗虫活性，从而降低农药的使用量，减少环境污染，具有重要的经济价值和应用前景。The present invention provides a new Bt protein Cry59Ba1 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 An amino acid sequence of one or more amino acids with 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 C ry59Ba1, its encoding gene and application

Technical field

The present invention relates to biological technical field, be specifically related to a kind of new Bt albumen and encoding gene and application.

Background technology

In the human being's production process, insect pest is the important factor that causes the agriculture production loss and influence human health.In order to reduce these losses, for many years, generally adopt the chemical prevention means to prevent and treat to crop pests and mosquito, but because the long-term, a large amount of of chemical pesticide use, caused the pollution to environment, pesticide residue increases in the agricultural byproducts, has brought harm for human existence and health.In addition, chemical pesticide has has also killed and wounded natural enemy and other useful thing in kill pests, destroyed the eubiosis.Compare with chemical prevention, biological control has safe, effective, lasting characteristics.And a series of problems of having avoided chemical prevention to bring.Therefore, biological control technology has become the focus of people's researchs.In biotic pesticide, bacillus thuringiensis is the quasi-microorganism sterilant that purposes is the widest in the world, output is maximum at present.

Bacillus thuringiensis (Bacillus thuringiensis, be called for short Bt) be a kind of gram positive bacterium, its distribution is very extensive, when forming, gemma can form the parasporal crystal of being formed by protein with insecticidal activity, have another name called insecticidal crystal protein (Insectididal crystal proteins is called for short ICPs), ICPs is by the cry genes encoding, sensitive insect there is strong toxicity, and to higher animal and people's nontoxicity.At present Bt has become the strong substitute of chemical synthetic pesticide in the control of agricultural pests, injurious forest-insect and sanitary insect pest, and Bt still be that transgenic pest-resistant engineered plant important function of gene is originated.

From strain HD-1, cloned since first can express the gene of insecticidal activity from Schnepf in 1981, people separating clone the gene of more than 500 kind of coded insect-killing crystallin, they are defined as different group, subgroup, class and subclass (Crickmore N respectively according to the amino acid sequence coded homology, et al.Microbiol Mol Biol Rev, 1998,62:807-813; Http:// www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/).Generally speaking, Cry1, toxalbumin such as Cry2 and Cry9 are effective to lepidoptera pest; Wherein the maximum of research are Cry1 and Cry9 proteinoid, and the insecticidal crystal protein molecular weight of their codings is 130-140kD, and many genes have been widely used in the control of the lepidoptera pest of plant at present.The toxin protein that Tribactur Israel subclass (B.thuringiensis subsp.israelensis is called for short Bti) produces has fine insecticidal activity to mosquito, is widely used in the control of mosquito.Simultaneously, Cyt albumen has cytolytic, the resistance that some Cry albumen is had synergism and delays insect.

The history in existing more than 50 year of Utilization of pesticides based on the Bt insecticidal crystal protein, at first never detect insect to the resistance of Bt, but, begin mid-term 80 year last century, resistance problem (the McGaughey that constantly in laboratory and field test, is confirmed, W.H.1985.Science.229:193-195), reason mainly is continue to use single variety and inferiorly cause the Bt of dosage and the application of Bt transgenic anti-insect plants causes insect population to be subjected to the selective pressure of sterilant for a long time.1985, McGaughey report warehouse grain pest Indian meal moth (Plodia interpunctella) under the selective pressure of Dipel (the commodity preparation of Bt subsp.kurstaik HD-1), bred for 15 generations after, resistance increases by 97 times; Under the high dosage selective pressure, resistance can increase by 250 times.Nineteen ninety, the small cabbage moth that confirms big Tanaka in Hawaii has first produced tangible resistance (Tabashnik to the Bt sterilant, B.E., et al.1994.Proc.Natl.Acad.Sci.USA.91:4120-4124), since the nineties in last century, used ground such as long Shenzhen and Guangzhou of Bt sterilant time, Shanghai in China, find that the Bt sterilant obviously descends to the small cabbage moth prevention effect, mean that resistance forms (Feng Xia .1996. insect journal, 39 (3): 238-244; Hofte, H., 1988.Appl.Environ.Microbiol.54:2010-2017).Find in the laboratory at present and the field has at least tens kinds of insects that Bt and insecticidal crystal protein thereof have been produced resistance, arrive with the selective pressure mathematical model prediction, under the condition of Bt transgenic anti-insect plants selective pressure, insect will produce resistance (Schnepf, E., et al.1998.Mol.Biol.Rev.65 (3): 775-806).In addition, there are some researches prove that Bti does not find resistance problem as yet in the use in land for growing field crops, but mosquito constantly is confirmed in the laboratory to its resistance problem, this situation also may (Georghiou G P occur big Tanaka, 1997.Applied and Environmental Microbiology, 63:1095-1101.).

Be the loss of avoiding resistant insects to cause, seeking new high virulence Bt genetic resources is the effective way that addresses this problem, and this biological control to China has very important meaning.

Summary of the invention

First purpose of the present invention is to provide a kind of new Bt virulence protein resource at above-mentioned deficiency.

Second purpose of the present invention is to provide the gene of encoding said proteins.

The 3rd purpose of the present invention is to provide the application of above-mentioned albumen and gene.

The present invention separates the new bacterial strain BM59-2 of bacillus thuringiensis that obtains from the soil of Muchuan, Sichuan Province virgin forest area, this bacterial strain (is called for short CGMCC on January 12nd, 2009 at China Committee for Culture Collection of Microorganisms common micro-organisms center, address: No. 3, A, DaTun Road, Chaoyang District, BeiJing City, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, classification called after bacillus thuringiensis (Bacillus thuringiensis), preserving number is CGMCC No.2871.This bacterial strain is open in Chinese invention patent CN101531981B.

By the virulence test shows to BM59-2, the lepidoptera pest of BM59-2, Diptera pest etc. all have high virulence.Find that from BM59-2 genome and plasmid sequencing result there is a cry59 gene in this bacterial strain, further design its full-length gene primer, the clone obtains the cry59Ba1 gene, its nucleotide sequence is shown in sequence table SEQ ID No.1, total length is 2025bp, analysis revealed, GC content are 35.56%, the albumen that 674 amino acid of encoding are formed.After measured, its aminoacid sequence is shown in SEQ ID No.2.Adopt bacterial sigma7.0 promoter program that complete sequence is predicted in the softberry website and show, contain the sequence in RNA polymerase activation site in the gene coding region upstream, with its called after Cry59Ba1.The amino acid of Cry59Ba1 albumen is formed as table 1.

The amino acid of table 1Cry59Ba1 albumen is formed

Amino acid	Per-cent %	Amino acid	Per-cent %
				Ala(A)：	5.93	Met(M)：	1.78
Cys(C)：	0.59	Asn(N)：	6.68

Asp(D)：	4.60	Pro(P)：	5.04
				Glu(E)：	4.75	Gln(Q)：	3.86
Phe(F)：	3.56	Arg(R)：	3.71
				Gly(G)：	7.12	Ser(S)：	8.61
His(H)：	1.78	Thr(T)：	7.72
				Ile(I)：	7.86	Val(V)：	5.79
Lys(K)：	4.90	Trp(W)：	1.63
				Leu(L)：	8.46	Tyr(Y)：	5.64

Be to be understood that, those skilled in the art can be according to the aminoacid sequence (SEQ ID No.2) of PROTEIN C ry59Ba1 disclosed by the invention, do not influencing under its active prerequisite, replacing, lack and/or increase one or several amino acid, obtaining the mutant nucleotide sequence of described albumen.For example at nonactive section, the 29th Ala is replaced with Val, with the 15th Ala disappearance, the 664th is increased a Gly or increase an Ile, do not influence its activity.Therefore, Bt PROTEIN C ry59Ba1 of the present invention comprises that also aminoacid sequence shown in the SEQ ID No.2 is substituted, replaces and/or increases one or several amino acid, has the protein of being derived and being obtained by Cry59Ba1 with isoreactivity with Bt PROTEIN C ry59Ba1.

Gene of the present invention comprises the nucleotide sequence of encoding said proteins Cry59Ba1.

The invention provides the gene of the above-mentioned Bt albumen cry59Ba1 of coding, its nucleotides sequence is classified as:

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

(2) nucleotide sequence shown in the SEQ ID No.1 is substituted, lacks and/or increases one or several Nucleotide.

In addition, should be understood that the degeneracy of considering codon and the preferences of different plant species codon, those skilled in the art can use as required and be fit to the codon that specific species are expressed.

Gene of the present invention can be cloned or separate from Bt bacterial strain BM59-2 with protein and be obtained, and perhaps obtains by DNA or the synthetic method of peptide.

Gene of the present invention can be operably connected with expression vector, obtain to express the recombinant expression vector of albumen of the present invention, and then can pass through such as transgenic methods such as agrobacterium-mediated transformation, particle bombardment, pollen tube passage methods, described expression vector is imported the host, obtain changeing the transformant of cry59Ba1 gene, for example plant such as farm crop or fruit tree makes it possess anti-insect activity.

In one embodiment of the invention, the acquisition of Bt PROTEIN C ry59Ba1 recombinant expression vector is to obtain recombinant expression vector pET-59Ba by the cry59Ba1 gene being inserted into last structure of expression vector pET-28a (+).

In addition, can also obtain containing the fermented liquid of Cry59Ba1 albumen by fermentation bacterial strain BM59-2 of the present invention, it is prepared into sterilant, be used for the control of crop pests.Those skilled in the art can also be with said gene transform bacteria or fungi, by large scale fermentation production Bt albumen of the present invention.

The present invention also provides the application of Bt PROTEIN C ry59Ba1 in improving plant resistance to insect.

The invention provides the application of Bt PROTEIN C ry59Ba1 in cultivating transgenic plant.

Those skilled in the art can also with farm crop such as its converting cotton, corn, paddy rice, vegetables, make it possess corresponding anti-insect activity according to cry59Ba1 gene disclosed by the invention.For example: the degeneracy of utilizing codon, the gene order that the cry59Ba1 gene design is had paddy rice preference codon, again synthetic cry59Ba1 gene order is connected with carrier pCAMBIA1300, be transferred in the rice genome by agriculture bacillus mediated, thereby obtain having the transgenic paddy rice kind of anti-insect activity.

The invention provides Cry59Ba1 albumen is a kind of new Bt albumen, has insecticidal activity preferably, uses it for the preparation transgenic plant, can the specific killing insect, and reduce the usage quantity of agricultural chemicals, and reduce cost, reduce environmental pollution.Also do not have at present insect or insect to the report of this albumen generation resistance, therefore, Bt PROTEIN C ry59Ba1 of the present invention has important economic value and application prospect, is fit to large-scale application in the insect-resistance that improves plant.

Description of drawings

Fig. 1 shows is the gel electrophoresis figure of the cry59Ba1 full-length gene that obtains of clone, M wherein, DNAmarker; 1, cry59Ba1 gene;

Fig. 2 shows is that the enzyme of recombinant plasmid pET-59Ba1 is cut the evaluation collection of illustrative plates, wherein 1, and the DNA of insertion; 2, the Sac I+Not I double digestion product of recombinant plasmid pET-59Ba; 3, pET28 vector plasmid, M, DNA marker;

What Fig. 3 showed is that the SDS-PAGE that the cry59Ba1 gene is expressed in E.coli BL21 (DE3) detects figure; M wherein, albumen marker; 1, contain E.coli BL21 (DE3) expressing protein (negative control) of carrier pET-28a; 2, the E.coli BL21 (DE3) that contains recombinant plasmid pET-59Ba1 induces cellular lysate liquid without IPTG; 3, contain E.coli BL21 (DE3) cellular lysate liquid after IPTG induces of recombinant plasmid pET-59Ba1.

Embodiment

Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.

If do not specialize, biochemical reagents used among the embodiment are the commercial reagent, the conventional means that used technique means is well known to those skilled in the art among the embodiment.

The clone of embodiment 1cry59Ba1 gene

The present invention separates the new bacterial strain of the bacillus thuringiensis (Bacillus thuringiensis) that obtains from the soil of Muchuan, Sichuan Province virgin forest area, this bacterial strain on January 12nd, 2009 at China Committee for Culture Collection of Microorganisms common micro-organisms center (address: No. 3, A, DaTun Road, Chaoyang District, BeiJing City, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, classification called after bacillus thuringiensis (Bacillus thuringiensis), preserving number is CGMCC No.2871.

This example is cloned the full length sequence that obtains the cry59Ba1 gene by the following method.

Adopt genomic dna purification kit (available from match Parkson company) to extract total DNA of bacterial strain BM59-2 as the template of amplification cry59Ba1 gene, the design primer sequence is as follows:

P1：5’-ATGAATTCATATGAAAATAA-3’；

P2：5’-TTAATTAACAAATAAACCATT-3’

25 μ l PCR reaction systems:

Thermal cycle reaction: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 50s, 54 ℃ of 50s, 72 ℃ are extended 2min, 30 circulations; 72 ℃ are extended 10min; 4 ℃ of stopped reaction.The amplified reaction product is electrophoresis on 1% sepharose, puts and observes the pcr amplification result in the gel imaging system.The result has obtained being about the sequence of 2025bp by amplification as shown in Figure 1, and this sequence is checked order, and its nucleotide sequence is shown in SEQ ID No.1, and is consistent with aim sequence.

The acquisition of embodiment 2cry59Ba1 albumen

According to cyt1Da1 gene open reading frame two terminal sequences, design and synthesize a pair of special primer 59BaF:5 '-CG GAGCTCATGAATTCATATGAAAATAA-3 ' 59BaR:5 '-ATTTG CGGCCGCTTAATTAACAAATAAACCATT-3 ', 5 ' end primer underscore part base is respectively Sac I and Not I restriction enzyme site.Be that template increases with the total DNA of BM59-2, carrier pET-28a (+) after enzyme is cut product and carried out double digestion equally is connected, Transformed E .coli DH5 α competent cell, extract its plasmid enzyme restriction electrophoresis and verified that insertion segment size meets (Fig. 2) after the intended purposes fragment, changes recipient bacterium E.coli.BL21 (DE3) (buying in the Beijing Quanshijin Biotechnology Co., Ltd) again over to.With recombinant plasmid called after pET-59Ba, contain the recon called after E.coli.BL21 (59Ba) of recombinant plasmid.Positive transformant in the LB substratum, in 200r/min, 37 ℃ of incubated overnight, is transferred to the ratio of nutrient solution according to 1: 100 in the 1L triangular flask that contains 400mL LB nutrient solution again, and 200r/min, 37 ℃ of cultivations are as the OD of nutrient solution ₆₀₀When value reaches 0.6-0.8, add 0.6mmol/L IPTG and carry out abduction delivering 12h, centrifugation medium is collected thalline, abandons supernatant, adds 30mL 10mmol/L Tris-HCl (pH 8.0) ultrasonic disruption, detects with the expressing protein of SDS-PAGE.

In the precipitation of SDS-PAGE analysis revealed expression of gene product after the thalline ultrasonication (Fig. 3), molecular weight is about about 75kDa, conforms to the molecular weight of albumen of prediction.

Embodiment 3Cry59Ba1 albumen insecticidal activity assay

The cry59Ba1 albumen that embodiment 2 is obtained carries out insecticidal activity assay to yellow-fever mosquito.At first Cry59Ba1 albumen is mixed with 0.1,4,8,16,6 different concentration gradients such as 32,64mg/mL, every processing drops into 20 albopictus larvaes then, every processing repeats for 3 times, and E.coli.BL21 (DE3) and empty carrier pET-28a (+) are as negative control, and clear water is blank; Statistics behind the 12h, LC ₅₀Use the SPSS10.0 software analysis.

The result shows (table 1): the insecticidal activity that expression product is certain to the yellow-fever mosquito tool, LC ₅₀Be 46.32 μ g/mL.Give birth to survey the result and show, E.coli.BL21 (DE3) and empty carrier pET-28a (+), blank are not had an insecticidal activity to yellow-fever mosquito.

The insecticidal activity of the yellow-fever mosquito of table 1cry59Ba1

Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims

1. A Bt protein Cry59Ba1, its amino acid sequence is: the amino acid sequence shown in SEQ ID No.2.

2. A gene encoding the protein of claim 1.

3. the gene as claimed in claim 2, its nucleotide sequence is as shown in SEQ ID No.1 in the sequence listing.

4. The recombinant expression vector containing the gene of claim 2 or 3.

5. A host cell transformed with the expression vector according to claim 4.

6. The host cell of claim 5, which is a plant host cell.

7. A pesticide containing the protein of claim 1.

8. The application of the protein or its coding gene according to claim 1 in the preparation of insecticides, and the insecticides have insecticidal activity against Aedes mosquitoes.

9. Application of the protein or its coding gene according to claim 1 in cultivating transgenic plants.

10. The application of the protein or its coding gene as claimed in claim 1 in improving plant insect resistance, and the insect is Aedes mosquito.