Dissertation
Dissertation > Agricultural Sciences > Plant Protection > A variety of control methods > Biological control > The use of microbial pathogens

Screening, Identification of Bacillus Thuringiensis and Cloning of cry 1Ab Gene

Author LiFengMei
Tutor LiPing
School Sichuan Agricultural University
Course Biochemistry and Molecular Biology
Keywords Bacillus thuringiensis Insecticidal PCR cloning Dot blot hybridization
CLC S476.1
Type Master's thesis
Year 2003
Downloads 493
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Bacillus thuringiensis and Bacillus thuringiensis is an insecticidal microorganisms caused by insect resistance to insecticidal effect is obvious, and human, animal and pest natural enemies with little or no toxic effects, do not pollute the environment, and to preserve the ecological balance is not easy. characteristics such research is very important to carry out the screening, identification, molecular biology, fermentation of the novel Bacillus thuringiensis resources. Sichuan ecological conditions of Bacillus thuringiensis resource surveys and studies for novel insecticidal gene cloning and transgenic insect resistance breeding, the novel insecticidal engineering strains build and lay the foundation for the development and application of Bacillus thuringiensis preparations. Screened from the soil of Sichuan Wenjiang nearly 279 strains of Bacillus thuringiensis, combined with field control tests Pieris, rice shell insects, rice stem borer pests, screened strains of a variety of pests with a strong cytotoxicity Bacillus thuringiensis strain, its classification and identification, laboratory virulence tests, the separation and purification of insecticidal crystal proteins and related gene cloning research. In addition, this study also strains promoting ability of rice and other biological characteristics been studied. Obtained the following results: 1. Collect soil samples 94, Wenjiang, Sichuan, sodium acetate - antibiotics were isolated Bacillus 279. Observed by morphological, physiological and biochemical identification, screened nine Bacillus thuringiensis: 39, D 3 H 12 D 1 , L < sub> 1 ,2,4-2 ,4-14, A 1 . 2. Insecticidal gene design universal primers and specific primers for PCR amplification. The results show that, inclusive of nine strains cry Ⅰ and cry Ⅴ the gene L 1 , 39 No. strains containing cry Ⅱ gene; 39,4-14,2 A 1 , D < sub> 3 strains containing cry Ⅲ gene. Each strain not only contain different gene type and contain the same genetic type in different genetic subtypes. 39,2,4-2,4-14 strains containing cry Ⅰ crylAb and crylAc genetic subtypes, and the rest of the strains containing crylAb, crylAc, and crylC three kinds cry Ⅰ class genetic subtype. 3. The biological characteristics of the strains, fermentation, to take fermentation products measured Pieris, rice shell insects, rice stem borer, diamondback moth and other indoor virulence. The results show that: the strains of vegetables Pieris best control effect, the average control effect of 90% or more; rice shell insects control efficiency up to 85.68%; rice stem borer control effect can reach up to 83.67%. From the various bacteria for pest control effect, 39, D 3 strains are high virulent strain. 4. A field test results show that: 9 strains of Pieris has good control effect, spraying the 72h of fermentation broth, the pests have reached the peak of death corrected mortality of 90% up to 100%. Prevention rice shell insects cell experiments, 6 days after spraying fermentation product, 39 strains corrected mortality was 85.06%, D 3 strain was 83.59%. 5 fermentation product spraying control of rice stem borer of rice, the results show that the number of dead heart of the rice is significantly less than the number of dead heart not spraying Bt bacteria rice comprehensive control effect, 39, D3 strains are highly toxic. Strains, consistent with the indoor test results. 5 biological characteristics of 39 strains, in line with its growth curve of bacteria typical growth curve characteristics. At pH 7, the culture temperature was 35 ℃, the largest growth, its control effect by ultraviolet impact. Baptist buds tests showed soaked through fermentation products, of which 39, A: the processed rice root length, stem length, root dry weight and stem dry weight was significantly higher than that of water and LB processing, and make well-developed root. The results of field experiments were sprayed with different concentrations of the different strains of rice panicle, seed setting rate, grain weight was significantly higher than that of the control. 7 No. 39 strain insecticidal crystal protein separation, purification, SDS GE electrophoretic display, a team, the No. 39 strain containing mainly three kinds of insecticidal crystal protein, molecular weight of 50kDa, 67kDa, 90kDao 8 using c processing IAb gene-specific primer pair No. 39 strain whole genome amplification, to obtain a gene fragment of 1330 decorated, the recovery, purification, ligation, transformation, and sequencing studies. Sequence analysis showed that the nucleotide acid fragment, the amino acid sequence in GenBank sipping IAb gene nucleotide and amino acid homology of more than 99%. 9 plasmid as a template, PCR product as a probe dot blot, cry1Ab, cry1Ac, crylC and crym gene located in the strains plasmid.

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