CN103421098A - Nematocide crystallin gene cry003-131 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microbe genetic engineering, and discloses separation and cloning of nematicidal crystallin gene originated from thuringiensis and biological activity of the nematicidal crystal protein gene on nematode. In the invention, a new insecticidal crystallin gene cry003-131 is obtained by being separated from wild-type thuringiensis Sbt003 (the preservation number in China Center for Type Culture Collection is CCTCC NO: M2012168); an encoded product of the gene is a new insecticidal crystallin Cry003-131; and the crystalline has high insecticidal activity on root-knot nematodes in south of China. The invention also comprises preparation of thuringiensis recombinant bacteria BMB1362 which expresses the insecticidal crystallin Cry003-131, the recombinant bacteria is preserved in China Center for Type Culture Collection and the preservation number is CCTCC NO: M2012058.

Description

Nematicide crystal protein gene cry003-131 and application

Technical field

The invention belongs to Strategies of Agricultural Bio-control and agriculture microbiological genetic engineering field.Be specifically related to one and there is the separation of the insecticidal crystalline gene of eelworm-killing activity, the application of albumen in microbial pesticide and genetically modified crops of cloning with and encoding.

Background technology

Tribactur (Bacillus thuringiensis, Bt) be that a class is distributed widely in the gram positive bacterium among environment, the born of the same parents of sprouting in the middle and later periods of growing at it can form, and when following sporulation, can produce a class various insects is had to the specially insecticidal crystal protein of cytotoxicity (Insecticidal Crystal Protein, ICPs), thereby be different from wax-like bacillus (Bacillus cereus, Bc) and Bacillus anthracis (Bacillus anthracis, Ba).Bt is obtained in 1901 from separating in the silkworm larva body by Japanese scholars first, and is Tribactur (explaining sub-ox, 1990) in definite designation in 1915, and the separated evaluation of 40,000 multi-strain bacteria strain has been arranged at present.Experimental results show that, Tribactur is to comprising lepidopteran, Diptera, Coleoptera, Hymenoptera, 500 various insects of the Insectas such as Homoptera have toxic action, in addition, it can also prevent and treat Protozoa effectively, Nemathelminthes, Platyhelminthes, plasmodium, schistosomicide, multiple insect pest (the Schnepf H E such as mite class, Crickmore N, Rie J V, Lereclus D, Baum J, Feitelson J, Zeigler D R, Dean D is thuringiensis and its pesticidal crystal proteins.Microbiol Mol Biol Rev H.1998.Bacillus, 62:775-806), therefore Tribactur biological control and the transgenic pest-resistant breeding with the agroforestry important pests that be widely used, and kept the safe handling record of nearly 90 years.

1981, Schnepf has separated first insecticidal crystalline gene first with Whiteley from Tribactur Kurstaki strain HD-1, after this people find and have identified a plurality of new killing genes successively, and it successfully is applied to the field of microbial pesticide and genetically modified crops, and therefore clone new killing gene is the focus in the Tribactur research field always.Along with increasing of new number gene, the scholars such as Crickmore have formally proposed the new separation system that the Tribactur insecticidal crystal protein is classified by amino acid sequence homology in 1996, stipulated naming rule and principle of classification.Wherein the cry gene must be from Tribactur, the killing gene of coding parasporal crystal albumen; The cyt gene is the parasporal crystal albumen that a kind of coding has dissolved cell activity, and with known coded Cyt albumen, the gene of obvious sequence similarity is arranged.The homology of the aminoacid sequence of deriving according to full-length gene is divided into four grades by insecticidal crystalline gene.Classification boundary line between level and level is homology 95%, 78% and 45% (Crickmore N, Zeigler DR, Feitelson J, Schnepf E, van Rie J, Lereclus D, Baum J, Dean DH.1998.Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.Microbiol.Mol.Biol.Rev.62:807-813).

Insecticidal crystal protein is the topmost insect killing substance of Bt bacterial strain, at present existing more than 200 the identified clone of cry gene.The cloning process of traditional cry gene mainly comprises: bioassay method, Southern hybrid method, protein sequencing method, build library method, various round pcrs etc., however the shortcomings such as that aforesaid method exists is time-consuming, effort and poor specificity.Doctor Ye Weixing of the state Key Laboratory of Agricultural Microbiology at applicant place utilizes s-generation high throughput sequencing technologies to set up the method for the novel cry gene of a kind of Rapid identification, thereby effectively overcome above-mentioned shortcoming, concrete grammar is referring to Fig. 1 and http://bcam.hzaubmb.org/BtToxin_scanner/.

The plant nematode disease be in a kind of world wide, generally occur by the caused Plant diseases of plant nematode.According to estimates, plant nematode causes the year rate of loss of global staple crops to be about 12.3%, annual direct economic loss is over 1,000 hundred million dollars, almost accounted for half in the loss caused whole Agricultural pests, be come the third-largest agriculture influence factor after Global climate change and the deterioration of the ecological environment (section imperial jade seal, Wu Gang. the plant nematode diseases control. Beijing: the Chinese agriculture science and technology .2002. of press).Root knot nematode is a kind of in plant nematode, it is settling down property of root system of plant endoparasitism biology, it is the important pathogen nematode of China cash crop, its host plant is over 3000 kinds, belong to 114 sections, wherein with the hazard of plant such as Solanaceae, Curcurbitaceae, Cruciferae particularly serious (Wang Laifa etc. the Biological Control of Root-Knot Nematodes progress. Nanjing Forestry University's journal (natural science edition), 2002,26 (1): 64-68.).For the method for preventing and treating the foundation nematode, have at present: Plant Quarantine, cultural control, chemical prevention, biological control.Plant Quarantine is a kind of good method that can effect a radical cure nematode, but its cost is high, is not suitable for large-scale promotion; The cultural control technology is in theory very effective, but has serious drawback in actual production; The chemical insecticide environmental pollution is serious, dangerous to people and animals in use procedure, many sterilants disabled (Liu Weizhi, plant pathogeny line insect is learned, Beijing: Chinese agriculture press, 2000).And compare with above method, biological control is stable owing to having, economical, long-acting and comparatively safe, can also remove the evil volume increase, pollution abatement, preserves the ecological environment simultaneously, is subject to gradually extensive concern.The eighties in last century, the people such as Bone confirmed that by research the Tribactur crystallin has insecticidal activity (Bone L W to nematode first, Bottjer K P, Gill S S.Trichostrongylus colubriformis:Egg lethality due to Bacillus thuringiensis crystal toxin.Exp.Parasitol., 1985,60:314-322.).Afterwards, a large amount of research work of U.S. Mycogen company have also further confirmed the effect (Bradfish G A (1992) .Process for controlling lepidopteran pests.EP19920920639.) of Tribactur parasporal crystal albumen to nematode.Simultaneously, doctor Guo Suxia of my chamber also clones and has obtained three new genes that root knot nematode has insecticidal activity.Yet, continuous popularization along with the use of Bt sterilant, various insects has produced resistance, in addition, existing insecticidal crystal protein still can not be prevented and treated some Agricultural pests effectively, therefore finding and clone how new insecticidal crystalline gene has become prevention and has controlled the critical path of target pest to Bt sterilant generation resistance, is the core content of various control strategies.Tribactur Sbt003 is the wild type strain by the agricultural microbiology National Key Laboratory preservation at applicant place, it can produce spherical, rhombus and long riziform parasporal crystal in sporulation, biological assay is tested and is shown, its crystallin has higher activity to beautiful rhabditis axei (Caenorhabditis elegans) and Meloidogyne incognita (Meloidgyne hapla).

Summary of the invention

The object of the invention is to clone and express a novel nematicide crystal protein gene cry003-131 from wild-type Tribactur Sbt003, the biological assay experiment shows that this albumen has high virulence to nematode, is indicating that this gene has good application prospect in research fields such as control root knot nematode pesticide preparation and insect-resistant transgenic crops.

Realize that technical scheme of the present invention is as described below:

The applicant analyzes and is cloned into new insecticidal crystalline gene cry003-148(details and refers to " embodiment " from the genomic information of wild-type Tribactur Sbt003).Through further order-checking discovery its coding region of cry003-131 of the present invention is by 3439 based compositions, its nucleotide sequence is as shown in SEQ ID NO:1.Through sequential analysis, find, the PROTEIN C ry003-131 of cry003-131 coding is comprised of 1179 amino acid, and the sequence of its protein is as shown in SEQ ID NO:2, and the predictive molecule size is 131kDa.The PROTEIN C ry003-131 that experimental results show that insecticidal crystalline gene cry003-131 coding by building Tribactur recombinant bacterium and biological assay has toxic action to Meloidogyne incognita.

Said gene sequence provided by the invention is a kind of new nematicide crystal protein gene, and this gene is with a wide range of applications in research fields such as control root knot nematode pesticide preparation and insect-resistant transgenic crops.

More detailed embodiment is referring to the description in embodiment.

The accompanying drawing explanation

Sequence table SEQ ID NO:1 is the sequence of the protein of the nucleotide sequence of Tribactur nematicide crystal protein gene of separating clone of the present invention and coding thereof.

Sequence table SEQ ID NO:2 is the sequence of protein of the Tribactur nematicide crystallin of separating clone of the present invention.

Fig. 1: the analysis strategy that is insecticidal crystalline gene cry003-131 in the embodiment of the present invention.

Fig. 2: the design of graphics and the physical map thereof that are cloning vector pEMB1412 in the embodiment of the present invention.

Fig. 3: the design of graphics and the physical map thereof that are shuttle expression carrier pBMB1422 in the embodiment of the present invention.

Fig. 4: be the SDS-PAGE electrophoretic analysis result of nematicide crystal protein gene cry003-131 expression and purification in recombinant bacterium BMB1362 in the embodiment of the present invention, in figure:

M: protein molecular weight standard;

1: insecticidal crystal protein Cry003-131 structure cell mixture albumen.

Specific embodiments

Below narration is embodiment according to embodiments of the present invention.Should be noted that embodiments of the invention only have illustration for the present invention, do not have restriction.About the standard operating instructions of the DNA that mentions in embodiment and the reagent that uses all can be referring to the method described in " molecular cloning experiment guide " (referring to Pehanorm Brooker and Russells, 2001, the molecular cloning experiment guide, the third edition, Jin Dongyan etc. (translating), Science Press, Beijing).Related other every experimental implementation in the present invention, be the ordinary skill in the art, the part that there is no special instruction in literary composition, those of ordinary skill in the art can be with reference to the present patent application various common tool books, scientific and technical literature or instructions book, handbook etc. a few days ago implemented.

The clone of insecticidal crystalline gene cry003-131 in embodiment 1 Tribactur Sbt003

The present invention delivered China on 05 15th, 2012 with this wild-type Tribactur of wild-type Tribactur Sbt003((Bacillus thuringiensis) Sbt003. Wuhan. and the center preservation of Wuhan University's Chinese Typical Representative culture collection, its preserving number is CCTCC NO:M2012168) as the nematicide crystal protein gene

The source bacterial strain, that this bacterial strain can produce in sporulation is spherical, rhombus and long riziform parasporal crystal, the biological assay experiment shows, its crystallin has higher activity to beautiful rhabditis axei and Meloidogyne incognita.

1. the extracting of the total DNA of Tribactur Sbt003

Adopt alkaline lysis commonly used to extract in a small amount.Its experimental procedure is as follows:

Wild-type Tribactur Sbt003 is spent the night in 28 ℃ of shaking tables activation, be forwarded in the LB liquid nutrient medium that 7mL is fresh by the inoculum size of 1/100 (v/v), in 28 ℃, with the 12000r/min concussion, is cultured to logarithmic growth mid-term; Cultured Sbt003 bacterium liquid is divided and is filled in the 1.5mL centrifuge tube, collect thalline (12000r/min, 1min), and with STE solution (10mM TrisHCl[pH8.0], 1mM EDTA[pH8.0], 100mMNaCl) wash 1 time; Above-mentioned thalline is suspended to 100 μ L solution I (50mM sucrose, 25mM TrisHCl[pH8.0], 10mM EDTA[pH8.0]), adds 20 μ L N,O-Diacetylmuramidases (50mg/mL) to mix, on ice standing 2 ~ 3h or spend the night; Add 200 μ L 2% sodium lauryl sulphate (SDS) solution, after light and slow mixing, in 55 ℃ of dry baths, process 30min; Add 100 μ L 5M NaCl solution, after light and slow mixing as on ice, standing 10min; With the centrifugal 5min of 12000r/min, draw in supernatant liquor to a new centrifuge tube, add respectively RNase and Proteinase K to final concentration 10 μ g/mL and 10 μ g/mL, bathe 30min as for 37 ℃ of water-bath temperature after mixing; With phenol/chloroform/primary isoamyl alcohol solution (volume ratio is 24:25:1, v/v/v) extracting 2 times; Draw supernatant, and add dehydrated alcohol or isopyknic Virahol of 2 times of volumes, after the standing 30min of room temperature or-20 ℃ of standing 2h, with 4 ℃ of centrifugal 5min of 12000r/min, abandon supernatant, precipitate 1 time by 70% washing with alcohol; Air drying precipitation, with 20 ~ 30 μ L TE solution (10mM TrisHCl[pH8.0], 1mMEDTA[pH8.0]) dissolution precipitation, be total DNA sample of preparation.

2. Tribactur Sbt003 genome sequence determination and the prediction of new type disinsection crystal protein gene

By above-mentioned, take out to such an extent that total DNA of Tribactur Sbt003 send Shenzhen Hua Da gene company limited to carry out genome sequencing and sequence assembly, according to the information exchange recorded, cross Bt-toxin forecasting software in the website of applicant place state Key Laboratory of Agricultural Microbiology predicted ( Http:// bcam.hzaubmb.org/BtToxin scanner/), obtain candidate's insecticidal crystalline gene.

3. the clone of insecticidal crystalline gene cry003-131 in Tribactur Sbt003

According to Tribactur Sbt003 genomic information and a pair of Auele Specific Primer of Bioinformatics Prediction consequence devised, the DNA sequence dna of this primer pair is as follows: 5Ac-LF:5 '-TGCATTCAATGCGCTAGGTAA-3 ', 5Ac-LR:5 '-GTTTGAAGGGACTAAGGTGATTTC-3 ', and take the total DNA of Tribactur Sbt003 and carry out pcr amplification to obtain target gene as template, system and the program of PCR reaction are as follows:

20 μ L reaction systems comprise: 2 μ L 10 * PCR reaction buffers, and 1.6 μ L dNTP, each 0.5 μ L Auele Specific Primer 5B-LF and 5B-LR (20mM), 0.2 μ L template, 0.2 μ L Ex-taq enzyme, add deionized water and be supplemented to 20 μ L.

PCR response procedures and parameter are: 94 ℃, and 5min, 1 circulation; 94 ℃, 30s, 52 ℃, 30s, 72 ℃, 3.5min, 30 circulations; 72 ℃, 10min, 1 circulation; 24 ℃, 5min, 1 circulation.

After the target fragment that above-mentioned amplification is obtained reclaims the recovery of test kit (purchased from Omega company product) purifying by PCR, carry out the T/A clone, be connected to carrier pMD18-T (purchased from precious biotechnology Dalian company limited, be Takara company product) upper, obtain the recombinant vectors pEMB1412 (seeing Fig. 2) that contains insecticidal crystalline gene cry003-131.Above-mentioned connection product is transformed to bacillus coli DH 5 alpha, the recon obtained is taken out to plasmid and enzyme is cut checking, will the correct recon sample presentation order-checking (examining order is completed by Beijing AudioCodes Bioisystech Co., Ltd) of checking.Sequencing result shows that the target fragment that above-mentioned amplification obtains contains just like the nucleotide sequence shown in sequence table SEQ ID NO.1, and the applicant is cry003-131 (gene of being named in the present invention, means with the italic lowercase) by this unnamed gene.By the biological software analysis, find, nucleotide sequence as shown in sequence table SEQ ID NO.1 of one section of this gene codified and corresponding aminoacid sequence, predict that its molecular weight is 131kDa, the applicant is by its called after Cry003-131 (in the present invention, the albumen of being named means with the roman capitalization).

4. the sequential analysis of insecticidal crystalline gene cry003-131 in Tribactur Sbt003

Find that through further sequential analysis its coding region of cry003-131 of the present invention is by 3439 based compositions, the PROTEIN C ry003-131 of its coding is comprised of 1327 amino acid, the predictive molecule size is 131kDa, carry out BlastP and analyze discovery on ncbi database, this aminoacid sequence and Cry5Ac albumen have the highest similarity, its consistence is 50%, according to the nomenclature mo of crystal protein gene (referring to document Crickmore N, Zeigler DR, Feitelson J, Schnepf E, van Rie J, Lereclus D, Baum J, Dean DH.1998.Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins.Microbiol.Mol.Biol.Rev.62:807-813), this gene is classified as the novel cry gene of II class.

Expression and purification and the biological activity determination of embodiment 2 nematicide crystal protein gene cry003-131 in recombinant bacterial strain BMB1362

1. the structure of nematicide crystal protein gene cry003-131 shuttle expression carrier

The recombinant plasmid pEMB1412 that contains the cry003-131 gene in embodiment 1 and shuttle expression carrier pHT304 are carried out respectively to SacI and SphI double digestion, then enzyme is cut to product and isolated target fragment by 1% sepharose, and reclaim test kit (Omega company product) by gel and reclaimed, utilize the T4 DNA ligase (purchased from precious biotechnology Dalian company limited, be Takara company product)) connected, thereby obtained the recombinant plasmid pBMB1421 (seeing Fig. 3) that contains the cry003-131 gene, above-mentioned connection product is transformed to bacillus coli DH 5 alpha, and recon is carried out to enzyme and cut checking, consistent with expected results, can determine and nematicide crystal protein gene cry003-131 has been inserted into to shuttle expression carrier pHT304(and see Fig. 3) in.

2. the Expression and purification of nematicide crystallin Cry003-131

(concrete grammar is referring to document D Peng for the mode that above-mentioned shuttle expression carrier pBMB1422 is transformed by electricity, Y Luo, S Guo, H Zeng, S Ju, Z Yu, M Sun.2009.Elaboration of an electroporation protocol for large plasmids and wild-type strains of Bacillus thuringiensis.Journal of Applied Microbiology.106 (6): 1849-58) import to expressive host bacterium Tribactur BMB171(He J, Shao X, Zheng H, Li M, Wang J, Zhang Q, Li L, Liu Z, Sun M, Wang S, Yu is genome sequence of Bacillus thuringiensis mutant strain BMB171.J Bacteriol.192 (15) Z.2010.Complete: 4074-5), obtained containing gene recombination bacterial strain BMB1362(is shown in Fig. 3).Concrete grammar is: by Tribactur BMB1362 spend the night the activation after, be transferred to 40mLICPM substratum (peptone 0.6%, glucose 0.5%, CaCO ₃0.1%, MgSO ₄7H ₂O 0.1%, K ₂HPO ₄0.05%, pH7.0) in, cultivate 32-48h in 28 ℃ of shaking tables, substantially come off to the brood cell.The centrifugal 2min of 4 ℃ of 12000r/min collect thalline, successively with 1M NaCl-10mMEDTA solution and ddH2O washing precipitation 3 times respectively, then to the structure cell mixture after washing, add appropriate 50mM Na ₂CO ₃(pH10.5, containing 3% beta-mercaptoethanol), fully mix, and in 37 ℃, processes 40min.12000r/min, 4 ℃ of centrifugal 2min collect supernatants, centrifugal twice, fully to remove brood cell and nourishing body cell., precipitate 4-5h on ice or spend the night near the crystallin iso-electric point by 4M NaAc-HAc solution (pH4.5) regulator solution pH value.12000r/min, 4 ℃ of centrifugal 10min collecting precipitations, and with aseptic deionized water washing 3 times, save backup in-80 ℃, before using with alkaline Heps solution dissolving.Final pure protein is carried out to the SDS-PAGE detection, result as shown in Figure 4, target protein size is substantially identical with the expection size, proves that nematicide crystallin Cry003-131 of the present invention is on Host Strains Tribactur BMB171(file is shown in) in obtained successful expression.The applicant is by above-mentioned recombinant bacterium called after restructuring Tribactur (Bacillus thuringiensis)

BMB1362, deliver it to China on March 6th, 2012. Wuhan. and Wuhan University's Chinese Typical Representative culture collection center (CCTCC) preservation, preserving number is CCTCCNO:M2012058.

3. nematicide crystallin Cry003-131 is to the bioactive mensuration of Meloidogyne incognita

The second instar larvae of Meloidogyne incognita of usining detects the biological activity of insecticidal proteins Cry003-131 to nematode as target organisms.Get the tomato root infected by Meloidogyne incognita, tomato root is rinsed well gently with tap water, from root, the Meloidogyne incognita pieces of an egg are peeled off out, with 0.5%NaClO, pieces of an egg are sterilized 2 times, and with deionized water wash 3 times, then be placed in 25 ℃ of incubator hatching 3-5 days, the Meloidogyne incognita hatched can be used as the target of biological assay.The biological assay experiment is to carry out in 96 orifice plates, add 40-50 nematode in every hole, (concrete grammar is referring to the minor congruence to give birth to the survey system and be 100 μ L, the foundation of Tribactur parasporal crystal Protein against Plant-parasitic Nematodes bioassay method and the screening of supper toxic strain, Journal of Agricultural Biotechnology, 2007(15): 867-871).3 concentration gradients are established in whole experiment, and each concentration is established 3 repetitions, use deionized water as negative control.Add up mortality ratio after 5 days, after mortality ratio is proofreaied and correct, be converted into the probability value, result shows that nematicide crystallin Cry003-131 of the present invention correction lethality rate to Meloidogyne incognita when concentration is 400 μ g/mL is 44.44%.

Above experimental result illustrates that crystallin Cry003-131 of the present invention has high virulence to Meloidogyne incognita, and this albumen provides a kind of new resource for the control root knot nematode.

Claims (7)

1. the application of Tribactur cry003-131 gene in preparing the nematicide crystallin, is characterized in that the nucleotide sequence of this gene is as shown in sequence table SEQ ID NO:1.

2. the restructuring Tribactur BMB1362 containing nematicide crystal protein gene cry003-131, it is deposited in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:M2012058.

3. application claimed in claim 1, is characterized in that, described application comprises the application in preparing microbial pesticide by the nematicide crystallin.

4. application claimed in claim 1, is characterized in that, described application comprises the application in transgenic microorganism or transgenic plant by the nematicide crystallin.

5. the application of restructuring Tribactur BMB1362 claimed in claim 2 in the control parasitic nematode.

6. the application of restructuring Tribactur BMB1362 claimed in claim 2 in preparing microbial pesticide.

7. the application of restructuring Tribactur BMB1362 claimed in claim 2 in transgenic microorganism or transgenic plant.

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