Isolation And Identification Of A Nematicidal Bacillus Thuringiensis And Its ICPs Gene Cloning

Plant-parasitic nematodes has become an important pest in agricultural production, the scope and variety of plant parasitic nematode continues to expand, has become one of the more serious diseases in agricultural production due to the development of modern agriculture. For limitations of agricultural control and chemical control, the use of biological control of plant parasitic nematode is became research focus.In this study, Bacillus thuringiensis (Bt) strain YC-10is target.the YC-10strain was purified and identified, and its fermentation conditions and medium components are screened. The cry gene which synthesis insectcidal crystal proteins (ICPs) is amplified by PCR from YC-10, further the cry gene is prokaryotic expressed in E. coli,then the enginerring bacteria are constructed which contain the recombinant plasmid cry-pQE30and efficient expression. The main results are as follows:1、A rod-shaped bacterium which is nematicide and produce spore was isolated from tobacco. It is purified by heat and double antibiotic, and it is identified16S rRNA sequence BLAST conclude that it belong to Bacillus sp..Base on this,the rob-shaped bacteria is proved to Bacillus thuringiensis compare its morphologyical characters and Physiological characters with two standard Bacillus thuringiensis strains,named YC-10.2, Based on the rod-shaped bacterium is Bacillus thuringiensis, then using Orthogonal Design and Response Surface Design screen the Bacillus thuringiensis YC-10’s flasking fermentation conditions and medium composition. The Orthogonal Design show that the shake flask fermentation conditions were:initial shake flask fermentation medium volume60ml/500ml initial medium pH of the8.0, inoculum volume4%,37℃for culture conditions,200r/min culture40h, its production of spore reach7.85×108cfu/ml. Further the medium components are reduced from8to4species, but the spore yield increased to10.5×108cfu/ml.3、The SDS-PAGE of ICPs found that the protein size is approximate50KDA. Mainwhile indoor bioassay shows that the ICPs from Bacillus thuringiensis strain YC-10have higher virulence to M. incognita. The results show that the ICPs kill M. incognita J2’s LC50is4.262mg/L which are treated96h. Indicates that the YC-10strain has a huge potential in the prevention and control of plant nematodes.4、The ICPs synthesis gene (cry gene) is cloned by common PCR.But this fragment is’n an complete ORF.So the5’end and3’end sequence are completed by TAIL-PCR,which use the Genomic DNA as a template, then a ORF of this cry gene is prokaryotic expressed,and the engineering bacteria are obtained which can express ICPs.