Cloning Of Cry Genes From Bacillus Thuringiensis And Study On Chitinases Of Bacillus Circulans CT14

Delta-endotoxins (5 -endotoxin, or Insecticidal Crystal Proteins, JCPs) from Bacillus thuringiensis (Bt) are highly toxic to Lepidoteran pests and other pests from many orders. Chitinases are used in plant disease control with its high antifungal activities. The cloning and expression of 6 -endotoxin genes and the characteristics of chitinases from Bacillus circulans have been studied in this article. 1. A wild-type Bt isolate COOS, is high toxic to diamond back moth (PlutelIa xylostella) and produce long-bipyramidal parasporal crystal. By using PCR products as probes, Southern blotting showed that a positive band located on 8.5kb plasmid DNA fragment digested with PstI. The 8.5kb fragment containing full-length crylAb gene was cloned, subcloned and sequenced. The gene has been registered in EMBL/GenBank (Accession number is AF254640) and named crylAbI3 as a novel cry gene by International Nomenclature Committee of Bt 6 -endotoxin gene. The Cry lAb protein deduced from the nucleotide sequence of the crylAbl3 gene composed of 1155 amino acids and its MW was 130.6kDa. The net charge of CrylAb was pH4.845. 2. The results from Bt isolates B-G-8 by PCR-RFLP analysis suggested that BG8 contained more than one kind of cry-type gene including a novel cry gene. The plasmid DNA of B-G-8 was digested partially with Sau3AI and 5- 7kb fragments were collected and inserted into pUCP 19, then transformed into JMIO7. All transformants were screened with PCR method. A 6.9kb DNA fragment containing a full-length crylA gene in a positive clone has been cloned, subcloned and sequenced. The homologous analysis suggested that it had the highest identity (82.8%) to crylAc within the first 3000bps. The result indicated the gene was a novel gene. The gene has been registered in EMBL/GenBank (Accession number is AF28 1866). The expressed product composed of 1182 amino acids, and the MW of the protein was 134 kDa and net charge was pH4.985. 3. An 8.5kb PstI DNA fragment was inserted into a broad-host-range vector pGM 1105 (Bt-Ecoli-P]) and a recombinant plasmid pGM8 1 was constructed. The pGM8 I was transformed into E. coli, Pesudomonas and acrystalliferous Bt strain cryB. The transformants were named ETG8I-J, ETGSI-S, PfFGSI, and BiotI8l respectively. SDS-PAGE analysis proved that the 130.6kDa protein had been expressed. Both PfTG8 1 and BiotI8 I were found insecticidal activity. Mortality of P xylostella in 48hr was 90% when PfTG8 1 culture was used. LC50 of BiotI8 1 against sensitive P xylostella (Beijing population) and resistant P xylostella (Hainan population) were 2.35 ii 1/mi and 3.35 i.?1/mi at 24hr respectively. The toxicity of BiotI8l to the 1st instar larvae cotton bell worm (H Armigera) was slightly higher than that of the original Bt strain COOS. On the other hand, the DNA fragment containing cry]A gene has been inserted into a shuttle vector pHT3 15 (Bt-E. coli). The transformants ETG59 and BiotIS9 were gained. A new kind of protein, MW 7OkDa, was found instead of 1 34kDa on SDS-PAGE. The growth speed of BiotI8l and BiotIS9 were slightly slower than that of the original strain COOS and BG8 separately, so did the endotoxin protein expression. However, the expressed products by BiotI8 I were nearly matched with COOS. 4. Using chitin-plates screened fifty-five samples from Beijing. Four kinds of bacteria] strains, 12 kinds of Streptomyces spp. Strains which can produce chitinases were isolated. The isolate CT14...