Study On Cloning And Expression Of Cry Gene And Cloning Of Replication Region Of Bacillus Thuringiensis

A Bacillus thuringiensis strain Bt25 with high insecticidal activity against several primary Lepidopteran pests in China was identified by PCR-RFLP system, and subsequently a novel crylAa gene was cloned from it by PCR techniques. The results of sequencing and structural analysis showed that the gene, named crylAa14 by International Nomenclature Committee of Bt 5 - endotoxin , was 3552 bp and the open reading frame encoded a 133.7 kDa protein with 1183 amino acids. The isoelectric point of CrylAa14 was pH 4.755. Between 918 and 1180 aa, 22-23 amino acids of CrylAal4 were different from 11 of all 13 CrylAa protein except Cry1Aa9 and Cry1Aa13, and especially four amino acids were additional, but they could be found in CrylAb protein. Recombinant plasmid, pBYBl, with full length cry1Aa14 gene was constructed and transformed into a Bt acrystalliferous mutant strain HD73 cry. Result of SDS- PAGE analysis indicated that the expression of the protein was strong in the receptor. Bioassay showed that the toxic protein appeared high insecticidal activity against Plutella xylotella larvea. LC50 was 0.633g/mL.A Bt-E.coli shuttle vector pHT315 was deleted its replication region of Bt ,then constructed a novel vector named pHT315-1 which composed a multiple cloning site,erythromycin and ampicillin-resistance marker and could only replicated in E.coli.Used pHT315-1,a 5273 bps DNA fragment carrying a novel Bt plasmid replicon was isolated and registered in GenBank as AY278324.Sequence analysis showed that there were at least three ORF(Open Reading Frame) in the cloned DNA encoding 501,333,183aas.ORFl had 98% identities with replicating related protein ORI43 of Bt strain HD263.The others were no homology to any published Bt replicating related protein.After continuous cultured for 70h at 30 C without antibiotic selecting press.the stability of plasmid carrying cloned replicon in Bt acrystalliferous mutant strain HD73 cry was more than 98%.And growth curve also showed that the novel replicon was stable and could replicate normally.