Cloning And Sequencing Of The AHLase Gene

Using potato to analysis the pathogeny. And knew that the dosage of the experiment is 50μl. Determine the number of the Bucillus sp. and Bacillus thuringiensis subsp. in the same OD, and the result is that in the same OD there are about same mubeT bacteria of that all. Then, use the potato to filter the bacterium, and we know that the Bacillus thuringienis var. aisawai Heimel ACCC10016 can control the pathogeny best. Using the Agrobacterium tumefaciens to test the Bucillus sp. and Bacillus thuringiensis subsp., but the result cannot be expressed due to all kinds of conductions. Mixed to culture the different bacteria in the same condition, then at last knew there are AHLase in the both Bucillus sp. Bt4 and Bacillus thuringiensis subsp. Shandongiensis ACCC10314. And when the ratio is 2:15 of the Bucillus sp. Bt4 and pathogeny, the pathogeny cannot cause the potato to rot. The ratio of Bacillus thuringiensis subsp. Shandongiensis ACCC10314 and the pathogeny is 3:15.Extracting the genome DNA of the Bucillus sp. and the vector isolatedly.The primers were designed based on the thesises that had be published. And PCR amplifications were performed with the genomic DNA of Bucillus sp. and Bacillus thuringiensis subsp., PCR fragments of about 800bp were obtained and named SSl and SS10, which was from Bt4 and Bacillus thuringiensis subsp. Shandongiensis ACCC10314. Reclamation the production of PCR fragment.Analysised the gene fragments, results show that the complete sequence legthe of corresponding PCR product from Bucillus sp. Bt4 and Bacillus thuringiensis subsp. Shandongiensis ACCC10314 are 753bp. Comparison of the report homologue genes showed high homologies of over 96% in the nucleotide sequence, and alive part is"106HFDH~199H~221D". Otherwise, comparison of the other Bacillus thuringiensis subsp. showed more high homologies of 98%. The center of alive catalyse is His119, His 188 and Asp210. Constrict the fusing gene pGEX-SS1 and pGEX-SS10, and inducement it with IPTG, purification, electrophoresis by agar, result showed that the size were 55kDa.