CN1337466A - Method of constructing pesticidal engineering bacteria of Bacillus thuringinsis - Google Patents
Method of constructing pesticidal engineering bacteria of Bacillus thuringinsis Download PDFInfo
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- CN1337466A CN1337466A CN 00115955 CN00115955A CN1337466A CN 1337466 A CN1337466 A CN 1337466A CN 00115955 CN00115955 CN 00115955 CN 00115955 A CN00115955 A CN 00115955A CN 1337466 A CN1337466 A CN 1337466A
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Abstract
The present invention relates to a method for constituting bacillus thuringiensis insecticidal engineering bactrium, which is characterized by that it utilizes PCR technique to clone two auxiliary protein genes of p19 and p20 form Israeli subspecies of thuringiensis spore, and utilizes gene engineering method to constitute engineering bacterium with specific virulence. As compared with existent technology it raises expression quantity of bacillus thuringiensis insecticidal crystal protein, promotes crystal formation, raises insecticidal efficiency and reduces production cost.
Description
The invention belongs to the construction process field of pesticidal engineering bacteria of Bacillus thuringinsis, relevant with the microbial pesticide gene engineering method.
Tribactur is a range of application microbial pesticide the most widely in the world at present.At first all be to use the wild type strain of screening to produce, along with development of molecular biology, people utilize engineered means that wild type strain is transformed gradually, utilize in conjunction with the method that shifts as Ecogen company, the 88Mda plasmid of coleopteron toxin gene cry3A gene changes HD-263-8 over to, the crylAc gene that resulting new bacterial strain EG2421 has the 88MDa plasmid that contains the cry3A gene and exists on the 60MDa plasmid that itself has with carrying extremely among the bacterial strain EG2158.The nineties, along with the maturation of electricimpulse technology, people can directly directly change the plasmid that contains goal gene over to wild type strain.Nineteen ninety-five, Ecogen register of company serve as the biotic pesticide RAVEN that produces bacterial strain with engineering bacteria EG7673, it contains multiple copied reorganization cry3Ba gene plasmid, can produce unusual high-caliber Cry3 insecticidal crystal protein.
At present, utilize mostly both at home and abroad and improve protein yield by the copy number that increases certain insecticidal crystalline gene, but test shows, the copy number of insecticidal crystalline gene has a max-thresholds in the Tribactur cell, surpass this value, proteic synthetic no longer increase, and the high virulence of Bacillus thuringiensis is former in various different cry gene synergism, and certain gene overexpression will suppress the normal expression of other gene.Utilize the effect of accessory protein can improve expression of gene, can not influence other expression of gene again at transcriptional level.Therefore, constructed engineering bacteria insecticide efficiency and the production cost of existing method all remains further to be improved.
The objective of the invention is to overcome the deficiency of prior art, utilize to help albumen to make up a kind of Su Yun gold brood cell disinsection engineering bacteria, to improve the expression amount of its insecticidal crystal protein, promotes crystal formationly, the raising insecticide efficiency reduces production costs.
The present invention implements by the following technical programs:
A kind of construction process of pesticidal engineering bacteria of Bacillus thuringinsis, its process comprises PCR, its step comprises:
1) from the Tribactur Israel subclass, clones the accessory protein gene of two of p19, p20 and the genotype one of wild type strain;
2) auxiliary gene is carried out digestion with restriction enzyme, and is connected to as on the shuttle plasmid or the carrier that dissociates, make auxiliary gene can be in Tribactur normal expression;
3) shuttle plasmid that will contain p19, p20 respectively electricity be transformed into wild type strain;
4) by plasmid map, PCR, Southern hybridization and polyacrylamide gel electrophoresis screening, evaluation transformant;
5) by shake-flask culture, the biological assay screening transformant that leavening property is good, virulence is high.The electric respectively method that is transformed into wild type strain of the shuttle plasmid of the shuttle plasmid of the described p19 of containing, p20 and p19, p20 gene and cry gene comprises:
With 272mM sucrose, the solution of 15% glycerine preparation at the mid-50-80 μ of 0.4cm electricimpulse cup l competent cell, adds plasmid 5 μ l (about 1 μ l) as damping fluid.Electricimpulse adopts 25 μ F, 200 Ω, and the condition electric shock of 6.25-9KV/cm is once.
Described screening and authentication step of carrying out transformant by plasmid map, PCR, Southern hybridization (DNA-DNA hybridization) and the gel electrophoresis of polyacrylamide nitrogen comprises:
With 1%D albumen, 0.5% yeast powder, 1%NaCl, the nutrient solution incubated overnight activated spawn of pH7 is again with activation bacterium liquid inoculation (1/100) LB nutrient solution.Treat thalli growth to mid-log phase, collect thalline, (10mM TrisCl, 1mMEDTA pH8.0) wash bacterium once to TE.Thalline is suspended from 100 μ l solution I (50mMGlucose, 25mM TrisCl (pH8.0), 10mM EDTA), adds 5-10 μ l N,O-Diacetylmuramidase (20mg/ml), and later operation is undertaken by the alkaline process plasmid extraction.
The method that described colibacillary plasmid extraction follows conventional lines.
The condition of described PCR is: 94 ℃ 1 minute, 49 ℃ 1 minute, 72 ℃ 1 minute, 25 circulations.
It is described that (the DNA-DNA hybridizing method comprises: the plasmid electrophoresis adopts 0.55% sepharose, and Hybond membrane uses nitrocellulose filter (Hybond), specifically changes film step and hybridization and colour developing according to a conventional method.
Described polyacrylamide gel electrophoresis method comprises: 200 μ l are centrifugal for L8 substratum fermented liquid, precipitation is respectively washed twice with 1MNaCl and sterilized water, throw out is suspended from the 1ml sterilized water again, getting 100 μ l mixes with sample buffering (2 *) on the equivalent, handled 3 minutes in the boiling water, get 10-20 μ 1 point sample electrophoresis, solution preparation and other concrete operations are according to a conventional method.
The step of described shake-flask culture, the biological assay screening transformant that leavening property is good, virulence is high comprises: biological assay culture medium prescription be: soybean cake powder 4%, W-Gum 2%, fish meal 1%, peptone 0.1%, yeast powder 0.2%, KH
2PO
40.1%, MgSO
47H
2O0.07%, CaCO
30.2%, this is L
8Substratum.
The living survey method of small cabbage moth and bollworm according to a conventional method.Beet armyworm gives birth to the survey method according to a conventional method.But be adjusted on the infection feed formulation, by Tegosept E, formaldehyde etc. change acetic acid (final concentration 0.005%) into sanitas.Change 72 hours observing time into.
Described strain fermentation culture medium prescription is: carbon source 5.0%, nitrogenous source A4.0%, nitrogenous source B2.0%, nitrogenous source C0.2%, somatomedin 0.1%, KH
2PO
40.2%, CaCO
30.2%.With above-mentioned substratum loading amount in the 20ml/500ml triangular flask, pH value 7.0, inoculum size 1%.
Described culture condition is: cultivate down at 30 ℃
Compare under identical conditions by above substratum and control medium, substratum fermentation titer of the present invention reaches more than the 4000IU/ μ l, and comparison has improved 8.7% according to substratum.
The present invention has following positive effect: (1) is because auxiliary gene can solve the high expression level problem of bacterial strain, the present invention utilizes auxiliary gene to make up a series of pesticidal engineering bacteria of Bacillus thuringinsis, as BMB21882, BMB15358, BMB83383, YBT803-1-19, show that invention is practical in the process that makes up engineering bacteria, good reproducibility.Through succeeding transfer culture, auxiliary gene can be in engineering bacteria stable existence, express normal, to the growth moment-less influence of recipient bacterium.
(2) control cotton and the efficient Tribactur engineering bacteria of the vegetables pest WG-001 that utilizes p20 to make up, its field control small cabbage moth effect improves 2.65 times than the product Dipel of U.S. Abbott company, improve 3.1 times than chemical pesticide 20% Belmark EC, prevention effect reaches 85%-90%.The prevention effect of bollworm is improved 20% than Dipel, and suitable with the virulence of 800 times of diluents of 40% acephatemet, preventive effect is at 80%-91%.
(3) the LC50 value of above-mentioned several different engineering bacteria all has significantly and improves.
Below in conjunction with chart and embodiment the present invention is further described: p19 helps the influence that albumen is expressed wild strain among the present invention:
Table 1 recipient bacterium YBT803-1 and transformant YBT803-1-19 are to the virulence of small cabbage moth
P20 helps the influence of albumen to wild type strain among the present invention:
| Bacterial strain | Regression equation y=a+bx | Correlation coefficient r | ????LC50/μg.L-1 |
| ?YBT803-1-19 | ??Y＝0.948＋2.126x | ??0.9786 | ????627.713 |
| ?YBT803-1 | ??Y＝0.292＋2.477x | ??0.983 | ????1648.588 |
The big or small bacterial strain of the natural bacterial strain of table 2 and pBMB1808 transformant parasporal crystal thereof/stain YBT1520 BMB21882 YBT1535 BMB15358 YBT833 BMB83383 length/Length 1.85 2.45 1.76 2.34 1.87 2.39 wide/2.31: 1 2.31: 1 2.35: 1 2.01: 1 2.34: 1 2.51: 1 volume/V 0.39 0.92 0.33 0.98 0.24 0.72 of Width 0.8 1.06 0.75 1.12 0.80 0.95 length/width L/W*30 crystal of every kind of sample random measurement; Unit of length is μ m, and volume unit is μ m
3 *PBMB1808 is the carrier that dissociates that contains the p20 gene.
*BMB21882, BMB15358, BMB83383 are respectively the transformants that contains pBMB1808 of wild type strain YBT1520, YBT1535, YBT833.
Natural bacterial strain of table 3 and pBMB1808 transformant thereof are to virulent strain/strain regression equation LC of small cabbage moth
50RYBT1520 y＝5.87＋1.43X 0.249 0.96BMB21882 y＝5.60＋0.84X 0.210 0.95YBT1535 y＝6.14＋1.72X 0.214 0.97BMB15358 y＝5.84＋1.32X 0.229 0.99YBT833 y＝6.80＋2.50X 0.247 0.95BMB83383 y＝5.66＋1.45X 0.339 0.92
*For the examination larva be three age small cabbage moth, be the brilliant mixtures of born of the same parents of washed PM culture for test agent.Add up mortality ratio after infecting 48h.LC
50Unit is μ l/ml, refers to the μ l number of contained fermented liquid among every ml.
Natural bacterial strain of table 4 and pBMB1808 transformant thereof are to virulence/strain regression equation LC of bollworm
50RYBT1520 y＝3.58＋1.14X 2.941 0.97BMB21882 y＝4.12＋0.56X 40.000 0.94YBT1535 y＝4.33＋1.71X 2.457 0.97BMB15358 y＝4.03＋0.57X 52.631 0.97YBT833 y＝4.28＋1.44X 3.086 0.94BMB83383 y＝4.00＋0.89X 12.500 0.91
*For just incubating bollworm, is the born of the same parents brilliant mixture of washed PM culture for test agent for the examination larva.Add up mortality ratio after infecting 72h.Repeat for three times; LC
50Unit is μ l/ml, refers to the μ l number of contained fermented liquid among every ml.
Claims (6)
1, a kind of method that makes up pesticidal engineering bacteria of Bacillus thuringinsis, its process comprises PCR, it is characterized in that described step comprises:
1) from the Tribactur Israel subclass, clones two accessory protein genes consistent of p19, p20 with the genotype of wild type strain;
2) auxiliary gene is carried out digestion with restriction enzyme, and is connected on the appropriate carriers, make auxiliary gene can be in Tribactur normal expression;
3) shuttle plasmid that will contain p19, p20 respectively electricity be transformed into wild type strain;
4) by plasmid map, PCR, DNA-DNA hybridization and polyacrylamide gel electrophoresis screening, evaluation transformant;
5) by shake-flask culture, the biological assay screening transformant that leavening property is good, virulence is high.
1, a kind of method that makes up pesticidal engineering bacteria of Bacillus thuringinsis according to claim 1 is characterized in that the described method of step 3) comprises:
With 272mM sucrose, the solution of 15% glycerine preparation at the mid-50-80 μ of 0.4cm electricimpulse cup l competent cell, adds plasmid 5 μ l as damping fluid, adopt at 25 μ F, and 200 Ω, electric shock is once under the electricimpulse condition of 6.25-9KV/cm.
3, a kind of method that makes up pesticidal engineering bacteria of Bacillus thuringinsis according to claim 1 is characterized in that the described method of step 4) comprises:
With 1%D albumen, 0.5% yeast powder, 1%NaCl, the nutrient solution incubated overnight activated spawn of pH7. is again with activation bacterium liquid inoculation (1/100) LB nutrient solution.Treat that thalli growth is to mid-log phase, collect thalline, TE (10mM TrisCl, 1mMEDTA pH8.0) washes bacterium once, and thalline is suspended from 100 μ l solution I (50mMGlucose, 25mM TrisCl (pH8.0), 10mM EDTA), add 5-10 μ l N,O-Diacetylmuramidase (20mg/ml), later operation is undertaken by the alkaline process plasmid extraction.
4, a kind of method that makes up pesticidal engineering bacteria of Bacillus thuringinsis according to claim 4 is characterized in that the condition of the PCR in this step is: 94 ℃ 1 minute, 49 ℃ 1 minute, 72 ℃ 1 minute, 25 circulations.
6, the construction process of according to claim a kind of pesticidal engineering bacteria of Bacillus thuringinsis, it is characterized in that the culture medium prescription that the biological assay in the described step 5) is adopted is: soybean cake powder 4, W-Gum 2%, fish meal 1%, peptone 0.1%, yeast powder 0.2, KH
2PO
40.1, MgSO
47H
2O0.07%, CaCO
30.2%.
7, a kind of method that makes up pesticidal engineering bacteria of Bacillus thuringinsis according to claim 1 is characterized in that the fermentative medium formula in the described step 5) is: carbon source 5.0%, nitrogenous source A4.0%, nitrogenous source B2.0%, nitrogenous source C0.2%, somatomedin 0.1%, KH
2PO
40.2%, CaCO
30.2%.
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|---|---|---|---|
| CN 00115955 CN1337466A (en) | 2000-08-15 | 2000-08-15 | Method of constructing pesticidal engineering bacteria of Bacillus thuringinsis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101347129B (en) * | 2008-09-01 | 2011-12-14 | 佛山市正典生物技术有限公司 | Bacillus thuringiensis suspending agent for killing mosquito |
| CN105219788A (en) * | 2015-10-25 | 2016-01-06 | 华中农业大学 | The application of the nematicide albumen NEL in Tribactur YBT1520 |
-
2000
- 2000-08-15 CN CN 00115955 patent/CN1337466A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101347129B (en) * | 2008-09-01 | 2011-12-14 | 佛山市正典生物技术有限公司 | Bacillus thuringiensis suspending agent for killing mosquito |
| CN105219788A (en) * | 2015-10-25 | 2016-01-06 | 华中农业大学 | The application of the nematicide albumen NEL in Tribactur YBT1520 |
| CN105219788B (en) * | 2015-10-25 | 2019-03-05 | 华中农业大学 | The application of nematicidal albumen NEL in thuringiensis YBT1520 |
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