CN103570811B - Bacillus thuringiensis gene cry1Ah3 and application thereof - Google Patents

Abstract

The invention relates to bacillus thuringiensis gene cry1Ah3 and application thereof, and belongs to the field of biological prevention and control. BT strain Bt S6 is cloned into a new gene named as cry1Ah3, wherein the nucleotide sequence of cry1Ah3 is shown as SEQ ID NO.1. The amino acid sequence of protein Cry1Ah3 coding the gene is shown as SEQ ID NO.2. According to indoor protein active detection, high activity on ostrinia nubilalis, cotton bollworm, rice-stem borer and plutella xylostella is achieved, and a new biological resource is provided for biological prevention and control.

Description

Bacillus thuringiensis Genes cry1Ah3 and application thereof

Technical field

The present invention relates to biological technical field, particularly relate to a kind of Bacillus thuringiensis Genes cry1Ah3 and its application.

Background technology

Insect pest is one of major reason causing crop production reduction, and the loss reducing insect pest is the important channel increasing grain and fodder crop output.The loss that world food and fodder crop ultimate production cause because of insect pest every year according to statistics reaches 14%, and the financial loss caused directly to agriculture production is up to hundreds billion of dollar.The loss paddy rice underproduction 10% that China causes because of insect pest every year, wheat yield 20%, the cotton underproduction more than 30% [Xia Qizhong, Zhang Mingju, anti-insect pest of the plant gene and application thereof, Ezhou college journal, 2005, (5): 56-60.].Employing sprays the means of prevention such as chemical pesticide and biotic pesticide and no doubt can alleviate insect causing harm to farm crop, but chemical pesticide causes environmental pollution, and biotic pesticide cost is higher.For a long time, spray chemical insecticide in a large number, not only can strengthen the resistance of insect, beneficial insect and other ecosystem are wrecked, and serious environment pollution, improve production cost, destroy the eubiosis.Therefore, reduce sterilant usage quantity, development modern plants resist technology, has become one of problem must faced in Agricultural Sustainable Development.

Bacillus thuringiensis (Bacillus thuringiensis, be called for short Bt) is a kind of widely distributed gram positive bacterium, is a kind of strong and to the avirulent entomopathogen of natural enemy to insect virulence, to higher animal and people's nontoxicity.It is that research is at present goed deep into the most, the most widely used microbial pesticide, has activity to 16 order 3000 various pests.Bt can form insecticidal crystal protein (Insecticidal CrystalProteins in the sporulation phase, ICPs), also delta-endotoxin (delta-endotoxin) is claimed, its shape, structure and size all with its virulence close relation [Schnepf.E, Crickmore.N, Van Rie.J., Lereclus.D, Baum.J, Feitelson.J, Zeigler.D.R., Dean.D.H.Bacillus thuringiensis and its pesticidal crystal proteins.Microbiol.Mol.Biol.Rev, 1998, 62 (3): 775-806.].

Clone first ICPs gene of Bt from Schnepf in 1981 etc., and in the aminoacid sequence of DNA base sequence and proteins encoded thereof that to have delivered it for 1985, ended in June, 2008 and found and cloned 412 kinds of ICPs genes.Tribactur insecticidal crystal protein is because of its good disinsection effect, safety, the advantage such as efficient and be widely used in pest control.

The routine transgenic anti-insect plants that beats the world for 1996 gets permission application in the U.S., and the gene that it uses is from Bt cry1Ac.In ensuing several years, turn the pest-resistant corn of cry1Ab gene, turn the pest-resistant potato of cry3Aa gene etc. and apart come out.In China, since starting the formal Insect Resistant Cotton promoted containing cry1Ac/cry1A gene from 1998, be widely planted.In genetically modified crops business-like first 12 years (1996-2007), owing to can obtain continual and steady income, peasant planting genetically modified crops amount increases year by year.2007, global genetically modified crops cultivated area rate of increase reached 12%, namely increased by 1,230 ten thousand hm2, reached 1.143 hundred million hm2 (2.824 hundred million acres).First 12 years, genetically modified crops commercialization all brings economy and environment benefit to the peasant of industrialized country and developing country.

Because the anti insect gene kind of current commercial insect-resistant transgenic crops is more single, there is insect sanctuary and reduce the risk risen with pest resistance to insecticide in spread plantation like this.Therefore that be constantly separated high virulence or the new incompatible risk avoiding pest resistance to insecticide to rise of genome is needed.

Therefore, screening and separating clones Bt killing gene that is new, high virulence, killing gene resource can be enriched, for genetically modified crops and engineering strain provide new gene source, improve the insect resistant effect of Bt transgenic product, and the resistance risk of insect to Bt toxalbumin can be reduced, avoid new eco-catastrophe to come, there are important economy, society and ecological benefits.

Summary of the invention

The invention provides a new Bacillus thuringiensis Genes cry1Ah3, its indoor protein-active detects, there is high reactivity to Pyrausta nubilalis (Hubern)., bollworm, rice-stem borer, small cabbage moth, and also have good activity to eating-core bean worm, for biological control provides new Biological resources.

A kind of insecticidal proteins, called after Cry1Ah3, its aminoacid sequence is as shown in SEQ ID NO2.

A kind of B. thuringiensis insecticidal gene, its nucleotide sequence coded above-mentioned insecticidal proteins Cry1Ah3.

Described killing gene called after cry1Ah3, its nucleotide sequence is as shown in SEQ ID NO1.

The application in kill pests of above-mentioned killing gene or albumen, described insect is Pyrausta nubilalis (Hubern)., bollworm, rice-stem borer, small cabbage moth or eating-core bean worm.

Described application is that albumen is used for described insect as the effective constituent of sterilant.

The present invention is GMCC3459 from BT bacterial strain Bt S6(deposit number) clone obtain a new gene, submit Bt NK called after cry1Ah3 to, through order-checking, its nucleotide sequence is as shown in SEQ ID NO1, and its aminoacid sequence is as shown in SEQ ID NO2.Carried out analysis of physical and chemical property with software to Cry1Ah3 albumen, this full length gene 3483bp, the amino acid of coding is 1160, calculates that coded molecular weight of albumen is about 131kDa.The theoretical iso-electric point of this albumen is 5.10, is slightly acidic protein.

Its indoor protein-active detects, and has high reactivity, for biological control provides new Biological resources to Pyrausta nubilalis (Hubern)., bollworm, rice-stem borer, small cabbage moth.

Accompanying drawing explanation

Figure 1B t S6 scanning electron microscopic picture

Fig. 2 Bt S6PCR product electrophoretogram

Fig. 3 cry1Ah3 gene and cry1Ah1 Gene sequence comparison

Fig. 4 Cry1Ah3 gene and Cry1Ah1 Gene sequence comparison

Fig. 5 cry1Ah3 gene expression analysis

Embodiment

Below in conjunction with embodiment, the present invention is described in further details.

Material described in following experiment is commercially available.

Embodiment 1

1. strain morphology observes characteristic:

Bt inoculation, in 1/2LB substratum, is cultivated about two days for 30 DEG C, after the release of microscopic examination brood cell crystal, scraping culture distillation washing 3-4 time, be suspended in 1mL sterilized water, spore crystalline substance mixing drop is on sheet glass, dry, fix through osmic acid, and after through alcohol serial dehydration, critical point drying, ion sputtering metal spraying (2nm), New Bio-TEMH-7500 scanning electron microscopic observation is taken pictures (figure), and result display Bt insecticidal proteins forms biconical crystal.

2. the cloned and sequenced of gene:

2.1 gene clones:

Design primer amplification full length gene:

1AH5 atggagatagtgaataatcagaatc

1AH3 ttcctccataaggagtaattccacgc

With the genome of Bt bacterial strain Bt S6 for template, surpass the new cry gene of fidelity dna polymeric enzymatic amplification with above-mentioned primer, Phusion, obtain the fragment of about 3.5kb.This full-length gene is cloned into pEB carrier.ContigExpress program in sequencing result Vector NTI Suite9 software package is spliced.Obtain the sequence of this gene, and translated into aminoacid sequence and see annex.

Detailed step is as follows:

1) Bt S6 genome preparation

By bacterial strain Bt S6 streak inoculation on LB substratum, 30 DEG C cultivate to brood cell release; On scraping flat board, whole thalline, in 1.5ml EP pipe, fully suspends with 100 μ l TE solution as far as possible; Add 100 μ l 4%SDS solution, fully mix; Add 200 μ l NaAc (3M, pH4.5), mixing, centrifugal 10 minutes of 12000rpm;

(2) collect supernatant, add equal-volume Virahol ,-20 DEG C leave standstill 20 minutes;

(3) centrifugal 10 minutes of 12000rpm, abandons supernatant;

(4) precipitate with the washes of absolute alcohol of 70%, room temperature is dried;

(5) use 100 μ l TE solubilize;-20 DEG C save backup.

2) PCR reaction system:

PCR reaction conditions: 94 DEG C of sex change 1 minute, 50 DEG C of annealing 1 minute, 72 DEG C extend 4 minutes, 30 circulations, last 72 DEG C of extensions 10 minutes.

3) PCR result 0.7% agarose gel carries out electrophoresis detection (figure).

Result shows, and primer can increase the gene band of typical 3.5kB of can increasing from bacterial strain Bt S6, may be used for further killing gene clone.

4) killing gene is connected with carrier

Carrier 0.1-0.2 μ g

Object sheet segment DNA 0.5-1.0 μ g

5 × connect damping fluid 2 μ L

T4DNA ligase enzyme 1 μ L

Supply volume to 10 μ L with ultrapure water, fully mix, 16 DEG C connect a 4h or 4 DEG C connection and spend the night.

5) killing gene connects product conversion

(1) get 200 μ l competent cells (JM109) to be connected product with 5 μ L and fully to mix, ice bath 30min.

(2) 42 DEG C of heat shock 1.5min, ice bath 3min.

(3) add 800 μ l LB substratum 37 DEG C and cultivate 45min.

(4) get 200 μ l coated plates, add corresponding microbiotic (penbritin), and IPTG, X-gal, 37 DEG C of cultivations.

The sequential analysis of 2.2 new gene

Submit this gene to Bt NK, be named as cry1Ah3, the albumen of its coding is Cry1Ah3.

Carried out analysis of physical and chemical property (table 1) with software to Cry1Ah3 albumen: this full length gene 3483bp, the amino acid of coding is 1160, calculates that coded molecular weight of albumen is about 131kDa.The theoretical iso-electric point of this albumen is 5.10, is slightly acidic protein.

Table 1 Cry1Ah3 albumen analysis of physical and chemical property

The amino acid composition analysis of table 2 Cry1Ah3 albumen

This gene order and cry1Ah1 are compared, result display gene has certain difference in length with on DNA sequence dna, as shown in Figure 3 simultaneously.

This gene coded protein sequence and Cry1Ah1 albumen are compared, result display gene also has certain difference in length with on DNA sequence dna, as shown in Figure 4 simultaneously.

3, gene is studied at expression in escherichia coli:

3.1 expression vectors build

Full-length gene is cloned into pEB carrier, and positive colony is seeded in LB substratum, 37 DEG C, 230rpm overnight incubation, extracts plasmid, transformation of E. coli Rosetta bacterial strain.Abduction delivering after positive recombinant is identified through sequencing again after cutting qualification through PCR, enzyme.

3.2 Primary structure

ZYP-5052 substratum: Tryptones 1.0%, yeast extract 0.5%, 0.05mol/L Na ₂hPO ₄, 0.05mol/LKH ₂pO ₄, 0.025mol/L (NH ₄) ₂sO ₄, 0.002mol/L MgSO ₄.

Lactose self-induction protein expression:

1) single bacterium colony activates 12h in LB liquid nutrient medium 37 DEG C, 220rpm;

2) 1% be inoculated in 200mLZYp-5052 substratum, add 4mL50x5052 simultaneously, 20 DEG C, 240rpm, 48h abduction delivering;

3) 8000rpm, 3min collected by centrifugation thalline, with 10mL20mmol/L TrisCl (pH8.0) suspension thalline;

4) broken thalline (ultrasonic disruption is complete) 5min;

5) 4 DEG C, 12000rpm, 10min collected by centrifugation supernatant and precipitation, carry out protein electrophoresis detection, the results are shown in Figure 5, and protein electrophoresis detected result display Cry1Ah3 gene successful expression about 130kD albumen, albumen is in soluble component.

4, insecticidal activity assay

Small cabbage moth: adopt leaf dipping method, cabbage leaves clear water is cleaned and dries, choose fresh and tender consistent cabbage leaves and be cut into the close bulk of size, in testing sample diluent, soak 10min, dry, put into raw survey bottle, every bottle graft 2-3 instar larvae 20, each process in triplicate, is incubated in 25 DEG C of biochemical cultivation cases, cultivate dead, the alive borer population of 48h " Invest, Then Investigate ", and observe larval feeding situation.

Rice-stem borer: take 5g artificial diet, adds 500 μ l testing sample diluents, fully mixes, in the feed culture dish of mixing in sterilizing culture dish.Each culture dish access rice-stem borer newly hatched larvae 24, often processes in triplicate, with clear water process in contrast.Place in 26 DEG C of illumination boxs and cultivate, cultivate 5 days " Invest, Then Investigate " mortality ratio.

Bollworm: take 5g artificial diet and put in a sterilizing culture dish, adds 500 μ L testing sample diluents, fully mixes, and is sub-packed in the 24 porocyte culture plates through sterilizing (5% dipped into formalin).Access bollworm newly hatched larvae gently with writing brush, every Kong Yitou, often process in triplicate, place in 25 DEG C of illumination boxs, cultivate dead, the alive borer population of investigation in 5 days.

Pyrausta nubilalis (Hubern).: take 30 grams of artificial diet and be positioned in culture dish, adds the testing sample of different amount respectively, fully mixes, be sub-packed in the culture dish of sterilizing, access newly hatched larvae gently with writing brush, 20, every ware, each repetition 3 times, places in 25 DEG C of incubators, 97 days investigation results.

Rice-stem borer: take 30 grams of artificial diet and be positioned in culture dish, add the testing sample of different amount respectively, abundant mixing, be sub-packed in the middle of the test tube of sterilizing, newly hatched larvae is accessed gently, often pipe 10, each repetition 3 times with writing brush, place in 25 DEG C of illumination boxs, 96 hours investigation results.

Eating-core bean worm: the Soybean Field (bearing pods) catch in field 3,000 adults (sex ration is close to 1:1) being put into the good net of in advance cover is raised, allows its natural spawning, for raw survey after hatching.The testing sample that green soya bean is diluting is soaked 10s, dries, put into the beanpod of strip off, then this beanpod is put into raw survey bottle, every bottle graft worm 15, each process repetition 3 times, cultivate 96 hours investigation eating-core bean worm death toll.

Result display Cry1Ah3 albumen has high reactivity to Pyrausta nubilalis (Hubern)., bollworm, rice-stem borer, small cabbage moth, and also has good activity (see table 4) to eating-core bean worm.

Table 3 Cry1Ah3 albumen is to Pyrausta nubilalis (Hubern)., bollworm, rice-stem borer, small cabbage moth insecticidal activity

Table 4 eating-core bean worm insecticidal activity

Claims (5)

1. an insecticidal proteins, called after Cry1Ah3, its aminoacid sequence is as shown in SEQ ID NO2.

2. a B. thuringiensis insecticidal gene, its nucleotide sequence coded insecticidal proteins Cry1Ah3 according to claim 1.

3. B. thuringiensis insecticidal gene according to claim 2, called after cry1Ah3, its nucleotide sequence is as shown in SEQ ID NO1.

4. the killing gene described in Claims 2 or 3 or the application of albumen according to claim 1 in kill pests, described insect is Pyrausta nubilalis (Hubern)., bollworm, rice-stem borer, small cabbage moth or eating-core bean worm.

5. applying described in claim 4, is that albumen according to claim 1 is used for described insect as the effective constituent of sterilant.