Cloning Of Novel Cry Genes And Analysising Of The Plasmids From Bacillus Thuringiensis Y41

Bacillus thuringiensis is the most widely used as insecticidal micriobe for its high toxin, safety and the other merits. B. thuringiensis is a spore-forming bacterium that forms a parasporal crystal during the stationary phase of its growth cycle. The activity of B. thuringiensis is attributed largely to the insecticidal crystal protein encoded by the cry genes. Insecticidal crystal protein is a good tool for agriculture and the cry gene encoding the toxin is a key source of genes for transgenic expression to provide pest resistance in plants now. Searching for novel strains against pests is important for increasing the toxicity and host range and might provide alternatives for potential problems associated with insect resistance.The main content of this paper is for the identification and clone of the novel cry genes form the strain Y41. The parasporal crystals of B. thuringiensis isolated Y41 were observed to be spherically shaped and SDS-PAGE analysis indicated that the toxins accumulating within the cells consisted of major proteins of 30, 66 and 140 kDa and several minor proteins. Plasmid profiling of the B. thuringiensis isolates Y41 and the reference strains was carried out. Agarose gel electrophoresis of plasmid DNA preparations revealed that Y41 carried obvious but fewer bands when compared with those of B. thuringiensis standard strain serovar kurstaki (HD1) and israelensis.A pair primer was designed from a group cry genes that selected by the toxin molecular weight and the shape of the crystals. Four novel cry genes fragment were cloned by the PCR-RFLP method from B. thuringiensis isolate Y41, which was isolated from Hainan Province. Five novel cry genes were cloned by using gene walking sequencing and short gun library sequencing. These five novel cry genes were deposited in GenBank and named by the B. thuringiensis delta-endotoxin nomenclature committee as toxin05, toxin01, toxin02, toxin03 and toxin04 respectively. Another novel cry gene fragment was identified by using fosmid library end-sequencing and this novel gene fragment have a 65% identity with cry50Aa. An express and clone vector pEB was constructed and expressed toxin01, toxin02, toxin03 and toxin04 successfully. Insecticidal activity of Y41 was determined and the result indicated that this strain have no toxicity to Plutella xylostella, Pyrrhalta aenescens Fairm, Phaedon brassicae Baly and Culex pipiens.Plasmid pY13536 from Y41 were sequenced successfully by"shot gun"method, and the related anlysis were finished.In this work, six cry genes were cloned by PCR-RFLP and fosmid library method. This work provides new approach for cry gene cloning and novel cry genes for pest biological control. The anlysis of the plasmid of Y41 will provide idea to tell why the toxin plasmid contains so many cry genes.