CN102532285A - Bt protein Cry2Ac-like and coding gene and application thereof - Google Patents

Abstract

The invention provides a novel Bt protein Cry2Ac-like and a coding gene thereof. The Bt protein has an amino acid sequence displayed by sequencer identification No.2 (SEQ ID No.2) or an amino acid sequence displayed by the SEQ ID No.2 that an amino acid sequence is replaced, lacked and/or increased with one or a plurality of amino acids and has the identical activity. The Bt protein Cry2Ac-like can be used for preparing Bt insecticides, and the gene for coding the Bt protein Cry2Ac-like can be transformed into cotton, corns, rice, vegetables and other crops, and therefore the crops are led to have corresponding insect resistance activity, use amount of pesticides is reduced, environment pollution is reduced, and the Bt protein Cry2Ac-like and the coding gene thereof have important economic value and application prospect.

Description

A kind of Bt PROTEIN C ry2Ac-like, its encoding sox and application

Technical field

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

Background technology

In the human being's production process, insect pest is the important factor that causes the agriculture prodn 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 safe, effective, persistent characteristics that biological control has with chemical prevention.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 present the widest, the maximum quasi-microorganism sterilant of output of purposes in the world.

Bacillus thuringiensis (Bacillus thuringiensis is called for short Bt) is a kind of gram positive bacterium, and its distribution is very extensive;, gemma can form the parasporal crystal of forming by protein when forming 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 confirmed as different crowd, 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 that their are encoded 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 the Utilization of pesticides that is the basis with the Bt insecticidal crystal protein; The initial resistance of insect that never detect to Bt; But, mid-term 80 year last century, the resistance problem (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 receive 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, confirm first that in Hawaii big Tanaka's small cabbage moth has produced tangible resistance (Tabashnik, B.E. to the Bt sterilant; Et al.1994.Proc.Natl.Acad.Sci.USA.91:4120-4124); Since the nineties in last century,, found that the Bt sterilant obviously descends to the small cabbage moth control effect on China Application of B t sterilant time long Shenzhen and Guangzhou, Shanghai and other places; Mean that resistance forms (Feng Xia .1996. insect journal, 39 (3): 238-244; Hofte, H., 1988.Appl.Environ.Microbiol.54:2010-2017).Find at present in the laboratory 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 occur big Tanaka (Georghiou GP, 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 crucial meaning.

Summary of the invention

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

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 JF21-1 of bacillus thuringiensis that obtains from the soil of Muchuan, Sichuan Province virgin forest area; This bacterial strain (is called for short CGMCC on August 11st, 2011 at China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: No. 3, Yard 1, BeiChen xi 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.5119.

Through the virulence test shows to JF21-1, JF21-1 all has high virulence to lepidoptera pest, Diptera pest or the like.Find that from JF21-1 genome and plasmid sequencing result there is a cry2 gene in this bacterial strain; Further its full-length gene primer of design is cloned and is obtained the cry2Ac-like gene, and its nucleotide sequence is shown in sequence table SEQ ID No.1; Total length is 2066bp; Analysis revealed, GC content are 36.91%, the albumen that 688 amino acid of encoding are formed.Through measuring, its aminoacid sequence is shown in SEQ ID No.2.Adopt bacterial sigma7.0promoter program that complete sequence is predicted in the softberry website and show, contain the sequence in RNA polymerase activation site at the gene coding region upper reaches, its called after Cry2Ac-like.The proteic amino acid of Cry2Ac-like is formed like table 1.

The proteic amino acid of table 1Cry2Ac-like is formed

Amino acid	Per-cent %	Amino acid	Per-cent %
				Ala(A)：	5.23	Met(M)：	2.18
Cys(C)：	0.44	Asn(N)：	6.54

Asp(D)：	5.52	Pro(P)：	4.80
				Glu(E)：	5.23	Gln(Q)：	4.80
Phe(F)：	3.78	Arg(R)：	3.92
				Gly(G)：	6.1	Ser(S)：	8.14
His(H)：	2.62	Thr(T)：	7.12
				Ile(I)：	7.56	Val(V)：	5.38
Lys(K)：	5.09	Trp(W)：	1.16
				Leu(L)：	9.16	Tyr(Y)：	5.23

Be to be understood that; Those skilled in the art can be according to the aminoacid sequence (SEQ ID No.2) of PROTEIN C ry2Ac-like disclosed by the invention; Do not influencing under its active prerequisite, replacing, lack and/or increase one or several amino acid, obtaining said proteic mutant nucleotide sequence.For example, the 36th Ala is replaced with Val,, the 37th is increased a Gly or increase an Ile, do not influence its activity the 51st Ala disappearance at nonactive section.Therefore; Bt PROTEIN C ry2Ac-like of the present invention also comprises aminoacid sequence shown in the SEQ ID No.2 through replacing, replace and/or increasing one or several amino acid, has the equal active protein of being derived and being obtained by Cry2Ac-like with Bt PROTEIN C ry2Ac-like.

Gene of the present invention comprises the nucleotide sequence of encoding said proteins Cry2Ac-like.

The invention provides the gene of the above-mentioned Bt PROTEIN C ry2Ac-like 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 through replacing, lack and/or increasing 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 JF21-1 with protein and obtained, and perhaps obtains through DNA or peptide synthetic method.

Can gene of the present invention be operably connected with expression vector; Obtain to express the proteic recombinant expression vector of the present invention; And then can said expression vector be imported host, the transformant that obtains changeing the cry2Ac-like gene through such as transgenic methods such as agrobacterium-mediated transformation, particle bombardment, pollen tube passage methods; 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 ry2Ac-like recombinant expression vector is to obtain recombinant expression vector pET-2L through the cry2Ac-like gene being inserted into last structure of expression vector pET-28a (+).

In addition, can also obtain containing the proteic fermented liquid of Cry2Ac-like, it is prepared into sterilant, be used for the control of crop pests through fermentation bacterial strain JF21-1 of the present invention.Those skilled in the art can also be with said gene transform bacteria or fungi, through large scale fermentation production Bt albumen of the present invention.

The present invention also provides the application of Bt PROTEIN C ry2Ac-like in improving plant resistance to insect.

The invention provides the application of Bt PROTEIN C ry2Ac-like 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 cry2Ac-like gene disclosed by the invention.For example: the degeneracy of utilizing codon; The gene order that the cry2Ac-like gene design is had paddy rice preference codon; Again synthetic cry2Ac-like gene order is connected with carrier pCAMBIA1300; Be transferred in the rice genome through agriculture bacillus mediated, thereby obtain having the transgenic paddy rice kind of anti-insect activity.

It is a kind of new Bt albumen that the present invention provides Cry2Ac-like 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 the insect report to this albumen generation resistance, therefore, Bt PROTEIN C ry2Ac-like 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 cry2Ac-like full-length gene that obtains of clone, M wherein, DNAmarker; 1, the cry2Ac-like gene;

Fig. 2 shows is that the enzyme of recombinant plasmid pET-2L 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-2L; 3, plasmid pET28a plasmid; M, DNAmarker;

What Fig. 3 showed is that the SDS-PAGE that the cry2Ac-like gene is expressed in E.coli BL21 (DE3) detects figure; M wherein, albumen marker; 1, contain E.coli BL21 (DE3) cellular lysate liquid after IPTG induces of recombinant plasmid pET-2L; 2, the E.coli BL21 (DE3) that contains recombinant plasmid pET-2L induces cellular lysate liquid without IPTG; 3, contain E.coli BL21 (DE3) expressing protein (negative control) of carrier pET-28a.

Embodiment

Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, 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 1cry2Ac-like gene

The present invention separates the new bacterial strain JF21-1 of the bacillus thuringiensis (Bacillus thuringiensis) that obtains from the soil of Muchuan, Sichuan Province virgin forest area; This bacterial strain on August 11st, 2011 at China Committee for Culture Collection of Microorganisms common micro-organisms center (address: No. 3, Yard 1, BeiChen xi 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.5119.

This example obtains the full length sequence of cry2Ac-like gene through following method clone.

Adopt genomic dna purification kit (available from match Parkson company) to extract the template of total DNA of bacterial strain JF21-1 as amplification cry2Ac-like gene, the design primer sequence is following:

P1：5’-ATGACGATGAAAACAAGGCA-3’；

P2：5’-CTATATAGA?A?ATGGGAGTTACA-3’

25 μ l PCR reaction systems:

Thermal cycle reaction: 94 ℃ of preparatory 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 is as shown in Figure 1, has obtained being about the sequence of 2066bp through amplification, and this sequence is checked order, and its nucleotide sequence is shown in SEQ ID No.1, and is consistent with aim sequence.

The proteic acquisition of embodiment 2Cry2Ac-like

According to cry2Ac-like gene ORFs two terminal sequences, design and synthesize a pair of special primer L2AcF:5 '-CG GAGCTCATGACGATGAAAACAAGGCA-3 ', L2AcR:5 '-ATT TG CGGCCGCCTATATAGAAATGGGAGTTACA-3 ', 5 ' end primer underscore part base is respectively Sac I and Not I restriction enzyme site.With the total DNA of JF21-1 is that template increases; 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 (Fig. 2) after the insertion segment size accord with expectation purpose fragment, change recipient bacterium E.coli.BL21 (DE3) (purchase) again in the Beijing Quanshijin Biotechnology Co., Ltd.With recombinant plasmid called after pET-2L, contain the recon called after E.coli.BL21 (2L) 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, with SDS-PAGE expressing protein is detected.

In the deposition of SDS-PAGE analysis revealed expression of gene product after the thalline ultrasonication (Fig. 3), the Cry2Ac-like molecular weight is about about 78kDa, conforms to the molecular weight of albumen of prediction.

Embodiment 3Cry2Ac-like albumen insecticidal activity assay

The Cry2Ac-like albumen that embodiment 2 is obtained carries out insecticidal activity assay to yellow-fever mosquito.At first Cry2Ac-like albumen is mixed with 0.1,4,8,16; 32,6 different concentration gradients such as 64mg/mL, every then processing drops into 20 albopictus larvaes; Every processing repeats for 3 times, and E.coli.BL21 (DE3) and empty carrier pET-28a (+) are as negative control, and clear water is a 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 36.54 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.

Table 1Cry2Ac-like is to the insecticidal activity of yellow-fever mosquito

Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.

Claims (10)

1. Bt PROTEIN C ry2Ac-like, its aminoacid sequence is:

1) aminoacid sequence shown in the SEQ ID No.2;

2) aminoacid sequence shown in the SEQ ID No.2 is through replacing, lack and/or increasing one or more amino acid and have equal active aminoacid sequence.

2. coding claim 1 said proteic gene.

3. gene as claimed in claim 2 is following 1) or 2):

1) its nucleotide sequence is shown in SEQ ID No.1 in the sequence table; Or

2) by nucleotide sequence shown in the SEQ ID No.1 through replacing one or several Nucleotide, the nucleotide sequence that obtains.

4. the recombinant expression vector that contains claim 2 or 3 said genes.

5. by the said expression vector transformed host cells of claim 4.

6. host cell as claimed in claim 5, it is a plant host cell.

7. contain the said proteic sterilant of claim 1.

8. the described albumen of claim 1 or its encoding sox application in the preparation sterilant

9. the described albumen of claim 1 or its encoding sox application in cultivating transgenic plant.

10. the described albumen of claim 1 or its encoding sox application in improving plant resistance to insect.

Images