Bacillus Thuringiensis Resource Diversity Analysis And Insecticidal Gene Discovery

Bacillus thuringiensis，gram positive bacteria, it can produce parasporal crystal during spore periodand play an important role in the pest control. The crystal mainly consist by the insecticidal crystalproteins which encode by cry genes, because the commercial value of Bt insecticidal gene, andexploring new toxin genes and protecting intellectual property rights is one of the important Bt resourceresearch content.As the intellectual property rights reflect the comprehensive competitiveness, our country at adisadvantage situation now. At present, most of Bt insecticidal gene patents were controlled by foreignagricultural biotechnology companies, and our country contoled less than60new gene with patents. Thedevelopment of high-throughput sequencing technologies provides new idea for insecticidal genemining and became the principal means to find new Bt insecticidal genes. But the sequencing price isrelative higher and the redundancy in Bt resource will be a big waste; and now there was no method canbe applied in strain comparison. Therefore, there is an urgent need.to set up an efficient method that cananalyzing and comparing large scale sample, removing redundant strains, to improve the insecticidalgene discovery efficiency. This thesis, beyond isolating1500strains Bt strains, setting up Bt diversityanalysis method, analyzing the strains diversity, removing redundant strains, sequencing screening90strains, and the results are:Isolation and analysis of Bt strains: The toxic activity evaluation results indicated that most strainshave highly toxicity against lepidoptera pests, in1500tested strains, there are1346strains toxic toHelicoverpa armigera,813strains toxic to Ostrinia furnacalis and only a few strains have toxic to leafbeetle, just42strains. Microscopic examination results shown that crystals are different in shape, size,and types between strains, most of them were rhombus, also contains spherical or irregular shapecrystals. Insecticidal crystal protein profiles analysis indicated that most strains expressing130kDprotein, and few of strains have100kD、60kD bands. With typical characteristic of Bt insecticidalcrystal proteins. Plasmid profile analysis indicated that the bands size and number is different betweenstrains and some strains’ plasmid profile are identical, this result indicated that they are redundant strainin the collection.Set up Bt stain diversity analysis method. Firstly, a pair degenerated primer were designed toamplify multi-cry genes. The fastPCR software analysis results showed that the primer set can amplifymore than30types insecticidal gene families. The following PCR results shown that the primer canwork but the amplicon bands produced were very week. Then, the primer set was improved by adding flank specific sequence, the new primer set were significantly improved the amplification efficiency.Furthermore, the primer concentrations and annealing temperature of PCR system were optimized; andthe testing of72different serotype standard Bt strains shown that the optimized PCR system canamplify expect bands from more than95%of the strains. The multiple genes amplification ability of theoptimized PCR system was evaluate by HD12that containing multiple genes, the results indicated thatit amplified9different cry gene fragments from the HD12strain. These results state that the optimizedPCR system with the basic requirements for Bt strains cry gene fingerprinting for its widelyamplification spectrum. In this thesis, Bt genome DNA extraction method also improved, the newmethod can get homogeneous quality genome DNA. The strains genome DNA were amplified by theoptimized PCR system and the PCR products were digested by Hinf I. the digested PCR products wereanalyzed by LabChip GX Electrophoresis system. Totally, in this thesis,888strains PCR-RFLP patternwere analyzed. The strains similarity and phylogenetic tree were constructed by PCR-RFLP patternsdigitize and computing. The results indicated that the method described here can effectively remove theredundant strain and yield valuable strains for next toxin gene finding.Screening insecticidal gene by sequencing of90Bt strains:90different strains sequence wereanalyzed by HiSeqTM2000and assembled by SOAPdenovo, then, the nsecticidal genes were predictby GeneMark, the insecticidal genes were screen out by blast software packge; and the NCBI patentdatabase were used to get rid of these already pattened genes. Finaly,21new insecticidal genes havebeen revealed in this study.In conclusion, a Bt strain diversity analysis method set up, and it can be used in removing theredundancy strains in collection, finally completed the “strain isolationg redundant removingtoxin gene sequencing screening” high throughput insecticidal gene screening platform construction.This work provides important guarantee to enhance competitiveness of China in the Bt resourcesdiscovery.