CN1327048A - A strain of recombined broad spectrum bacillus thuringiensis - Google Patents

Abstract

The recombined broad-spectrum bacillus thuringiensis may be used to kill both lepidoptepous and doleopterus pests and the strain LCj-12 has high stability, high efficiency and broad pesticidal spectrum. It is used to prepare pesticide with multiple pesticidal effects, and has low cost, high economic value, no harm to human body, animal and plant, no environmental pollution. It has been passed through relevant toxicity and safety estimation.

Description

One strain of recombined broad spectrum bacillus thuringiensis

The present invention relates to genus bacillus, more specifically relate to the recombined broad spectrum bacillus thuringiensis.

The alleged strain of recombined broad spectrum bacillus thuringiensis (Bacillusthuringiensis LCJ-12) of the present invention is by what genetic engineering means made up lepidopteran and coleopteran pest all to be had the bacterial strain of high virulence, not only killed lepidopterous insects but also killed the characteristics of coleopteron according to this bacterial strain, get lepidopteran (Lepidoptera), Coleoptera (Coleoptera), its code LCJ formed in first letter of bacterium (Jun), be that the comform virulence that screens in the multiple group of engineering bacteria is the highest because of this bacterium again, and be numbered a strain of 12, so with this recombined broad spectrum bacillus thuringiensis called after Bacillus thuringiensis LCJ-12.

Lepidopteran is the maximum insect of harm during China's agriculture forest and husbandry is produced, and coleopteran pest is only second to lepidoptera pest to the harm of agriculture forest and husbandry production, colorado potato bug invasion is found in area, Xinjiang of China Huocheng in 1991 first, and rapid spread also causes significant damage to farm crop.Therefore, construct Coleoptera and all effective recombined broad spectrum bacillus thuringiensis of lepidoptera pest and use it to produce novel pesticide and have urgency and realistic meaning.In the prior art, document " the Bacillus thuringiensis recon crystal proteins that contains binary toxin gene analyze and to the toxicity of mosquito larvae " (China, JOURNAL OF MICROBIOLOGY, 1999,19 (1): be to pass through electrotransformation 1-5), the recombinant plasmid pCW2 that will contain the Bacillus sphaericus binary toxin gene transforms the wild-type bacillus thuringiensis subsp israelensis, obtains the recombinant bacterial strain B+CW-1 that a strain contains binary toxin gene; It is intracellular toxin and cytA toxin that recombinant bacterial strain can be expressed binary toxin, and forms 2 classes and be positioned at the outer parasporal crystal of gemma spore adventitia.Document " structure of disinsection prophylaxis genetically engineered subtilis " (China, the biotechnology journal, 1999,15 (2): be carrier with subtilis-bacillus coli shuttle plasmid vector pHB201 and pRP22 respectively 215-220), by the competence method for transformation, bacillus thuringiensis HD-1 insecticidal protein gene cry1Ac is imported rice sheath blight disease biocontrol strain subtilis B916; The electrophoretic analysis of engineering strain plasmid enzyme restriction, the nucleic acid marking (Southern) is analyzed and desinsection biological activity determination result has confirmed the importing of cry1Ac gene and the effective expression in B916 thereof; Bacteria inhibition assay has proved that engineering bacteria has kept the good bacteriostatic activity of former wild type strain.Document " bacillus thuringiensis subsp israelensis 130000 dalton kill the expression of mosquito protein gene in subtilis " kills mosquito protein gene subclone to the pNQ122 carrier with bacillus thuringiensis subsp israelensis, by the subtilis protoplast transformation, obtain forward and reverse clone's of KMr-CMs; Protein immuning hybridization (Western-blotting) proves that extremely the mosquito protein gene expressed and have the immunocompetent mosquito protein that kills in subtilis, the expressed mosquito albumen that kills has mosquito activity extremely in experiment.Bacterial strain in the above-mentioned various document all has the characteristics of self, and specific insect is had good killing effect, but does not all possess the ability that can kill lepidoptera pest and coleopteran pest simultaneously.

The purpose of this invention is to provide and a kind ofly not only killed lepidopteran but also killed the broad spectrum bacillus thuringiensis LCJ-12 of coleopteran pest.This bacterial strain can efficiently express external source insecticidal crystal protein plasmagene cry1Ac and self insecticidal crystal protein plasmagene cry3A, possesses the ability that can kill lepidoptera pest and coleopteran pest simultaneously, has the insecticidal effect of stability and high efficiency and broad spectrum.

In order to achieve the above object, a strain of recombined broad spectrum bacillus thuringiensis of the present invention (Bacillus thuringiensis LCJ-12) has following characteristic: a, this bacterial strain contains bacillus thuringiensis insecticidal crystal protein gene cry1Ac that kills lepidopterous insects and the bacillus thuringiensis insecticidal crystal protein gene cry3A that kills coleopteron; B, this bacterial strain produce rhombus and two kinds of insecticidal crystal protein matter of square; C, this bacterial strain can kill lepidopterous insects simultaneously and kill coleopteron; D, this bacterial strain contain the recombinant plasmid pHT304/1Ac10 that shuttles back and forth.

The size of the above-mentioned recombinant plasmid pHT304/1Ac10 that shuttles back and forth is 10453 base pairs, and contain bacillus thuringiensis plasmid pHT1030 and intestinal bacteria (E.coli) plasmid pUC19 the replication origin sequence, kill cry1Ac gene, penbritin (Amp) resistance and erythromycin (Em) resistant maker gene of lepidopterous insects.

A strain of recombined broad spectrum bacillus thuringiensis of the present invention (Bacillus thuringiensisLCJ-12) can be by the electric shock transformation technology, the insecticidal crystal protein plasmagene cry1Ac that kills lepidoptera pest is changed in the bacillus thuringiensis recipient bacterium of efficient Coleoptera extremely, measure and virulence bioassay through erythromycin (Em) resistance screening, genetic stability, obtain recombinant bacterial strain LCJ-12.

A strain of recombined broad spectrum bacillus thuringiensis of the present invention can be prepared by following method:

A, employing one plant height are imitated the bacillus thuringiensis bacterial strain (in China's typical culture collection center preservation, preserving number is CCTCC NO:M201003) that kills coleopteron, and as F-strain, this bacterial strain contains the cry3A gene; Adopt the pHT304/1Ac10 recombinant plasmid, this recombinant plasmid contains the cry1Ac gene of lepidopterous insects extremely;

B, the LB inoculation of medium intestinal bacteria that contain 100 μ g/ml penbritins, 35-37 ℃ is acutely rocked and cultivated 8-12 hour, and bacterial precipitation is resuspended in the ice-cold solution I of 100 μ l, concuss adds 200 μ l solution II; Add the ice-cold solution III of 150 μ l,,, in-20 ℃ of deposit D NA, obtain intestinal bacteria recombinant plasmid pHT304/1Ac10 with the ethanol of 2 times of volume supernatant liquors with equivalent phenol-chloroform-primary isoamyl alcohol extracting supernatant liquor in centrifugal 10 minutes of 4 ℃ of following 12000g;

C, recipient bacterium is seeded in the LB substratum, 28-30 ℃ after wave and culture 8-10 hour, ice bath 20-30 minute, centrifugal 10 minutes of 4 ℃ of following 12000g, earlier wash twice, wash once with the electric shock damping fluid again, be suspended at last in the electric shock damping fluid with the electric shock elutriant, obtain recipient bacterium electric shock competent cell, put-20 ℃ of preservations;

D, pHT304/1Ac10 plasmid DNA and recipient bacterium competent cell suspension fully are mixed, get 200 μ l and inject electric shock conversion cup, shock by electricity once under 2.5KV with the intestinal bacteria conversion instrument (E.coli GenePulser) that shocks by electricity, the liquid that will shock by electricity changes in the 4mlLB substratum, 28-30 ℃ of vibration cultivated after 2 hours, coat on the LB flat board that contains 20 μ g/ml erythromycin, cultivated 3 days down for 28-30 ℃;

E, on 20 μ g/ml erythromycin resistant panel, select single bacterium colony, smear for microscopic examination, choosing the bacterium colony with bacillus thuringiensis feature goes down to posterity on the erythromycin flat board, to detect its stability, go down to posterity and virulence bioassay and PCR detection through the heredity in 20 generations, obtain virulence recombined broad spectrum bacillus thuringiensis the highest and that contain cry1Ac and cry3A gene, be a strain of recombined broad spectrum bacillus thuringiensis LCJ-12 of the present invention.

Described LB substratum is: 0.5% sodium-chlor (NaCl), and 0.5% yeast powder, 1.0% Tryptones the rest is water, pH7.2-7.4.

Described microbiotic adopts: the penbritin working concentration is 100 μ g/ml, and the erythromycin working concentration is 20 μ g/ml.

Described electric shock elutriant is: two (2-hydroxyethyl) piperazine-N '-2-(ethyl sulfonic acid) of 1mmol/L (pH7.0) N-, be called for short HEPES.

Described electric shock damping fluid is: 1mmol/L (pH7.0) HEPES, 10% glycerine.

Described solution I is: 50mmol/L glucose, 25mmol/L Tutofusin tris (pH8.0), 10mmol/L ethylenediamine tetraacetic acid (EDTA) (pH8.0).

Described solution II is: 0.2mol/mL sodium hydroxide, 1% sodium laurylsulfonate.

Described solution III is: 5mol/L potassium acetate, 2.5mol/L glacial acetic acid.

The used examination worm of described virulence bioassay is: just incubate bollworm (Helithis armigera)

Larva and 2 willow fleautiauxia armata in age (Playiodera versicolora) larvas.

Described PCR detects and is meant with Auele Specific Primer detection cry1Ac and cry3A gene;

The specific primer sequence that detects the cry1Ac gene is: TYIUNI2 5 ' ATCACTGAGTCGCTTCGCATGTTTGACTTTATC3 ' TYIIAC 5 ' TCACTTCCCATCGACATCTACC3 '

The specific primer sequence that detects the cry3A gene is: CoL1A 5 ' GTCCGCTGTATATTCAGGTG3 ' CoL2A 5 ' AGGTGCCAACTAACCATGTT3 '

A strain of recombined broad spectrum bacillus thuringiensis LCJ-12 of the present invention also has following feature: morphological specificity and biochemical characteristic LCJ-12 bacterial strain are shaft-like: wide (μ m) 1.2-1.8

Do not catch on long (μ m) 3.0-5.0 Gram-reaction+protoplasma the bead+gemma ellipse of look+

Circular-

Interior life or interior partially giving birth to+

Sporangium expands-pH value 4.3-5.6 growth temperature in parasporal crystal rhombus and the cubic bodily form mobility+catalase+facultative suspicion gas of the anaerobic growth bacterium V-P reaction+V-P meat soup (℃): the highest 40-45

Minimum 10-15 egg yolk reaction+in 0.001% lysozyme growth+in 7%NaCL growth+produce acid+arabinose at pH6.0 culture medium growth+glucose to produce acid-wood sugar and produce acid-mannose product acid-hydrolyzed starch+utilize lemon hydrochlorate+reductive NO 3-NO2+take off in 1 week phenylalanine-hemolytic test+RNA enzyme test+genotype cry1Ac, cry3A antibiotic resistance erythromycin (Em) resistance

Advantage of the present invention and effect are: reorganization bacillus thuringiensis LCJ-12 can efficiently express external source insecticidal crystal protein plasmagene cry1Ac and self insecticidal crystal protein plasmagene cry3A, has the insecticidal effect of stability and high efficiency and broad spectrum.Because recombined broad spectrum bacillus thuringiensis LCJ-12 desinsection scope has comprised lepidopteran and coleopteran pest, use the sterilant of its preparation to possess the multiple insecticidal properties that kills lepidoptera pest and coleopteran pest, production cost and use cost have been saved, thereby improved economic worth, reduced expenses for prevention and control.The security and the wild bacillus thuringiensis that have now proved recombined broad spectrum bacillus thuringiensis LCJ-12 are as good as, harmless, free from environmental pollution to people, animal and other animal and plant, and can improve agricultural product quality, good economy and ecological benefits are arranged, have good popularizing application prospect.

Embodiment

Carried out fermentation lab scale and pilot scale, microbial inoculum production, field test, demonstration and land for growing field crops application, quality standardization and safety testing with recombined broad spectrum bacillus thuringiensis LCJ-12, its quality has reached the primary standard of Ministry of Agriculture's regulation.Its acute toxicity and ecological security have been got permission Ministry of Agriculture's genetic engineering body pilot scale level security and have been estimated and the evaluation of open-air release level security.

Claims (12)

1, one strain of recombined broad spectrum bacillus thuringiensis (Bacillus thuringiensis LCJ-12), this bacterial strain not only can kill lepidopterous insects but also can kill coleopteron, get lepidopteran (Lepidoptera), Coleoptera (Coleoptera), its code LCJ formed in first letter of bacterium (Jun), be numbered 12 because of this bacterium again, so with its called after Bacillusthuringiensis LCJ-12, it is characterized in that this bacterial strain has following characteristic: a, this bacterial strain contains the bacillus thuringiensis insecticidal crystal protein gene cry1Ac and the bacillus thuringiensis insecticidal crystal protein gene cry3A that kills coleopteron of lepidopterous insects extremely; B, this bacterial strain produce rhombus and two kinds of insecticidal crystal protein matter of square; C, this bacterial strain can kill lepidopterous insects simultaneously and kill coleopteron; D, this bacterial strain contain the recombinant plasmid pHT304/1Ac10 that shuttles back and forth.

2, a strain of recombined broad spectrum bacillus thuringiensis according to claim 1, it is characterized in that, the size of the described recombinant plasmid pHT304/1Ac10 that shuttles back and forth is 10453 base pairs, and contain bacillus thuringiensis plasmid pHT1030 and intestinal bacteria (E.coli) plasmid pUC19 the replication origin sequence, kill cry1Ac gene, penbritin (Amp) resistance and erythromycin (Em) resistant maker gene of lepidopterous insects.

3, a strain of recombined broad spectrum bacillus thuringiensis according to claim 1 is characterized in that, this bacterial strain can be prepared by following method:

A, employing one plant height are imitated the bacillus thuringiensis bacterial strain (in China's typical culture collection center preservation, preserving number is CCTCC NO:M201003) that kills coleopteron, and as F-strain, this bacterial strain contains the cry3A gene; Adopt the pHT304/1Ac10 recombinant plasmid, this recombinant plasmid contains the cry1Ac gene of lepidopterous insects extremely;

B, containing the LB inoculation of medium intestinal bacteria of penbritin, 35-37 ℃ is acutely rocked and cultivated 8-12 hour, and bacterial precipitation is resuspended in the ice-cold solution I of 100 μ l, and concuss adds 200 μ l solution II; Add the ice-cold solution III of 150 μ l,,, in-20 ℃ of deposit D NA, obtain intestinal bacteria recombinant plasmid pHT304/1Ac10 with the ethanol of 2 times of volume supernatant liquors with equivalent phenol-chloroform-primary isoamyl alcohol extracting supernatant liquor in centrifugal 10 minutes of 4 ℃ of following 12000g;

C, recipient bacterium is seeded in the LB substratum, 28-30 ℃ after wave and culture 8-10 hour, ice bath 20-30 minute, centrifugal 10 minutes of 4 ℃ of following 12000g, earlier wash twice, wash once with the electric shock damping fluid again, be suspended at last in the electric shock damping fluid with the electric shock elutriant, obtain recipient bacterium electric shock competent cell, put-20 ℃ of preservations;

D, pHT304/1Ac10 plasmid DNA and recipient bacterium competent cell suspension fully are mixed, get 200 μ l and inject electric shock conversion cup, shock by electricity once under 2.5KV with the intestinal bacteria conversion instrument (E.coli GenePulser) that shocks by electricity, the liquid that will shock by electricity changes in the 4mlLB substratum, 28-30 ℃ of vibration cultivated after 2 hours, coat on the LB flat board that contains erythromycin 20 μ g/ml, cultivated 3 days down for 28-30 ℃;

E, on erythromycin (20 μ g/ml) resistant panel, select single bacterium colony, smear for microscopic examination, choosing the bacterium colony with bacillus thuringiensis feature goes down to posterity on the erythromycin flat board, to detect its stability, go down to posterity and virulence bioassay and PCR detection through the heredity in 20 generations, obtain virulence recombined broad spectrum bacillus thuringiensis the highest and that contain cry1Ac and cry3A gene, be a strain of recombined broad spectrum bacillus thuringiensis LCJ-12 of the present invention.

According to the described preparation method of claim 3, it is characterized in that 4, described LB substratum is: 0.5% sodium-chlor (NaCl), 0.5% yeast powder, 1.0% Tryptones the rest is water, pH7.2-7.4.

According to the described preparation method of claim 3, it is characterized in that 5, described microbiotic adopts: the penbritin working concentration is 100 μ g/ml, and the erythromycin working concentration is 20 μ g/ml.

According to the described preparation method of claim 3, it is characterized in that 6, described electric shock elutriant is: two (2-hydroxyethyl) piperazine-N '-2-(ethyl sulfonic acid) of 1mmol/L (pH7.0) N-, be called for short HEPES.

According to the described preparation method of claim 3, it is characterized in that 7, described electric shock damping fluid is: 1mmol/L (pH7.0) HEPES, 10% glycerine.

According to the described preparation method of claim 3, it is characterized in that 8, described solution I is: 50mmol/L glucose, 25mmol/L Tutofusin tris (pH8.0), 10mmol/L ethylenediamine tetraacetic acid (EDTA) (pH8.0).

According to the described preparation method of claim 3, it is characterized in that 9, described solution II is: 0.2mol/mL sodium hydroxide, 1% sodium laurylsulfonate.

According to the described preparation method of claim 3, it is characterized in that 10, described solution III is: 5mol/L potassium acetate, 2.5mol/L glacial acetic acid.

According to the described preparation method of claim 3, it is characterized in that 11, the used examination worm of described virulence bioassay is: just incubate bollworm (Helithis armigera) larva and 2 willow fleautiauxia armata in age (Playiodera versicolora) larvas.

According to the described preparation method of claim 3, it is characterized in that 12, described PCR detects and is meant with Auele Specific Primer detection cry1Ac and cry3A gene;

The specific primer sequence that detects the cry1Ac gene is: TYIUNI2 5 ' ATCACTGAGTCGCTTCGCATGTTTGACTTTATC3 ' TYIIAC 5 ' TCACTTCCCATCGACATCTACC3 '

The specific primer sequence that detects the cry3A gene is: CoL1A 5 ' GTCCGCTGTATATTCAGGTG3 ' CoL2A 5 ' AGGTGCCAACTAACCATGTT3 '