CN103570811A - 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 with and application.

Background technology

Insect pest is one of major reason causing crop production reduction, and the loss that reduces insect pest is the important channel that increases grain and fodder crop output.Global grain and fodder crop ultimate production are every year because the loss that insect pest causes reaches 14% according to statistics, and the financial loss directly causing to agriculture production is up to hundreds billion of dollars.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 no doubt can alleviate insect to the causing harm of farm crop, but chemical pesticide causes environmental pollution, and biotic pesticide cost is higher.For a long time, spray in a large number chemical insecticide, 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 the problem that must face 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 higher animal and people's nontoxicity to insect virulence.It is that research is at present goed deep into the most, the most widely used microbial pesticide, and 16 order 3000 various pests are had to activity.Bt can form insecticidal crystal protein (Insecticidal CrystalProteins in the sporulation phase, ICPs), also claim delta-endotoxin (delta-endotoxin), 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.].

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

The routine transgenic anti-insect plants that beats the world for 1996 is got 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 appearances apart such as pest-resistant potato of cry3Aa gene.In China, since 1998 start formally to promote the Insect Resistant Cotton that contains cry1Ac/cry1A gene, be widely planted.In genetically modified crops business-like first 12 years (1996-2007), owing to obtaining 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%, increases 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 brought 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, so spread plantation exists insect sanctuary to reduce the risk rising with pest resistance to insecticide.Therefore need constantly the incompatible risk of avoiding pest resistance to insecticide to rise of genome separated high virulence or new.

Therefore, screening and separating clone Bt killing gene new, high virulence, can enrich killing gene resource, for genetically modified crops and engineering strain provide new gene source, improve the pest-resistant effect of Bt transgenic product, and can reduce the resistance risk of insect to Bt toxalbumin, 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, Pyrausta nubilalis (Hubern)., bollworm, rice-stem borer, small cabbage moth are had to high reactivity, and eating-core bean worm is also had to good activity, for biological control provides new Biological resources.

, called after Cry1Ah3, its aminoacid sequence is as shown in the SEQ ID NO2.

A killing 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 effective constituent using albumen as sterilant is for described insect.

The present invention is GMCC3459 from BT bacterial strain Bt S6(deposit number) clone obtained a new gene, submit the called after cry1Ah3 of Bt NK 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.With software, Cry1Ah3 albumen has been carried out to analysis of physical and chemical property, 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 Pyrausta nubilalis (Hubern)., bollworm, rice-stem borer, small cabbage moth is had to high reactivity, for biological control provides new Biological resources.

Accompanying drawing explanation

Figure 1B t S6 scanning electron microscope 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 is learned and is observed characteristic:

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

2. the clone of gene and sequential analysis:

2.1 gene clones:

Design primer amplification full length gene:

1AH5 atggagatagtgaataatcagaatc

1AH3 ttcctccataaggagtaattccacgc

The genome of Bt bacterial strain Bt S6 of take is template, with above-mentioned primer, the new cry gene of the super fidelity dna polymeric enzymatic amplification of Phusion, obtains the fragment of about 3.5kb.This full-length gene is cloned into pEB carrier.Sequencing result splices by the ContigExpress program in Vector NTI Suite9 software package.Obtain the sequence of this gene, and translated into aminoacid sequence and see appendix.

Detailed step is as follows:

1) Bt S6 genome preparation

By bacterial strain Bt S6 streak inoculation on LB substratum, 30 ℃ of cultivations discharge to brood cell; On scraping flat board, whole thalline, in 1.5ml EP pipe, fully suspend 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), mix, centrifugal 10 minutes of 12000rpm;

(2) collect supernatant, add equal-volume Virahol ,-20 ℃ standing 20 minutes;

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

(4) use 70% dehydrated alcohol washing and precipitating, room temperature is dried;

(5) with 100 μ l TE solution, dissolve;-20 ℃ save backup.

2) PCR reaction system:

PCR reaction conditions: 94 ℃ of sex change 1 minute, 50 ℃ of annealing 1 minute, 72 ℃ are extended 4 minutes, 30 circulations, last 72 ℃ are extended 10 minutes.

3) PCR result is carried out electrophoresis detection (figure) with 0.7% agarose gel.

Result shows, can increase from the bacterial strain Bt S6 gene band of the typical 3.5kB that can increase of primer can be cloned for further killing gene.

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 * connection damping fluid, 2 μ L

T4DNA ligase enzyme 1 μ L

With ultrapure water, supply volume to 10 μ L, fully mix, 16 ℃ of connection 4h or 4 ℃ of connections are spent the night.

5) killing gene connects product conversion

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

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

(3) add 37 ℃ of 800 μ l LB substratum to cultivate 45min.

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

The sequential analysis of 2.2 new genes

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

With software, Cry1Ah3 albumen has been carried out to analysis of physical and chemical property (table 1): 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 shows that 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 shows that 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, and 37 ℃, 230rpm overnight incubation, extracts plasmid, transforms intestinal bacteria Rosetta bacterial strain.Abduction delivering after sequencing is identified positive recombinant again after PCR, enzyme are cut evaluation.

3.2 gene induced expression

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 37 ℃ of LB liquid nutrient mediums, 220rpm;

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

3) the centrifugal collection thalline of 8000rpm, 3min, with 10mL20mmol/L TrisCl (pH8.0) suspension thalline;

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

5) 4 ℃, the centrifugal collection supernatant of 12000rpm, 10min and precipitation, carry out protein electrophoresis detection, the results are shown in Figure 5, protein electrophoresis detected result has shown Cry1Ah3 gene successful expression about 130kD albumen, and albumen is in soluble component.

4, insecticidal activity assay

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

Rice-stem borer: take 5g artificial diet, add 500 μ l testing sample diluents, fully mix in sterilizing culture dish, in the feed culture dish mixing.24 of each culture dish access rice-stem borer newly hatched larvaes, every processing in triplicate, is processed in contrast with clear water.Place in 26 ℃ 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, add 500 μ L testing sample diluents, fully mix, be sub-packed in 24 porocyte culture plates of sterilization (5% dipped into formalin).With writing brush, access gently bollworm newly hatched larvae, every Kong Yitou, every processing in triplicate, is placed in 25 ℃ of illumination boxs, cultivates 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, add respectively the testing sample of different amounts, fully mix, be sub-packed in the culture dish of sterilizing, with writing brush, access gently newly hatched larvae, 20, every ware, each repetition 3 times, places in 25 ℃ of incubators 97 days investigation results.

Rice-stem borer: take 30 grams of artificial diet and be positioned in culture dish, the testing sample that adds respectively different amounts, fully mix, be sub-packed in the middle of the test tube of sterilizing, with writing brush, access gently newly hatched larvae, 10 of every pipes repeat 3 times at every turn, place in 25 ℃ of illumination boxs 96 hours investigation results.

Eating-core bean worm: 3,000 adults catching in field (male and female ratio approaches 1:1) are put into the Soybean Field (bearing pods) that prior cover nets well and raise, allow its natural spawning, survey for raw after hatching.Green soya bean is soaked to 10s at the testing sample having diluted, dry, put into the beanpod of strip off, then this beanpod is put into the raw bottle of surveying, 15 of every bottle graft worms, each is processed and repeats 3 times, cultivates 96 hours investigation eating-core bean worm death toll.

Result shows that Cry1Ah3 albumen has high reactivity to Pyrausta nubilalis (Hubern)., bollworm, rice-stem borer, small cabbage moth, and eating-core bean worm is also had to good activity (in Table 4).

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 bacillus thuringiensis killing gene, its nucleotide sequence coded insecticidal proteins Cry1Ah3 claimed in claim 1.

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

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

5. application described in claim 4 is that effective constituent using albumen claimed in claim 1 as sterilant is for described insect.

Images