Studies On Gene Resources Of Bacillus Thuringiensis From Zhougong Moutain In Ya'an And The Cloning Of Insecticidal Crystal Protein Gene

In this study,A total of 132 Bacillus thuringiensis isolates were screened by Sodium acetate—anti—biotic method from 320 soil samples collected from the primeval forest in Mountt Zhong Gong of Sichuan province,and their average isolated rate was 11%. Preliminary study showed that several shapes of parasporal crystal such as bipyramidal, quadratend spherical shape were observed by microscopic observation in the B.thuringiensis isolates.It showed the great diversity of Bt strains in Mt.Zhong Gong. Therefore,we had preliminarily studied on the gene resources of Bacillus thuringiensis from Zhougong Moutain,Ya'an,and had cloned partial insecticidal crystal protein gene. The main results were as follows:1 The features of Bt distribution had been summarized on the base of analysis on elevation,resources and climate of 350 soil samples collected:the Significance of the Difference showed the isolation rate among different elevation under 1742 meter altitude. The Bt isolation rate of humus soil was higher than clay soil,and the clay soil higher than sandy soil.The Bt strains were more abundant in these area which had high vegetation coverage and good ecological conditions.The thicker the detritus layer was,the higher the Bt isolation rate was.The Bt isolation rate of sunny slope of mountains was also higher than the shady slope.2 The SDS-PAGE assay of parasporal crystal showed that Bt Strains which are toxic for Lepidoptera,coleopteran and espeasialy the dipteran insects were the most abundant in our collection,which shows the specificity of B.thuringiensis resourse.3 The cry gene-types of these 132 B.thuringiensis isolates were identified by use of PCR-RFLP.The results showed that 74 isolates harbored cryl genes,34 isolates cry2 genes, 13 isolates cry3 genes,8 isolates cry4 genes,16 isolates cry7 genes,2 isolates cry8 genes,16 isolates cry9 genes,7 isolates cry30 genes,2 isolates cry40 genes.46 isolates harbored unidentified genes,which account for 34.8%.This results demonstrated that the cryl gene is the most abundant,the cry2 gene the more,and the cry9 and cry3 gene the abundant.4 The cry9Ea gene had been cloned(Accession numbers was EU760456).Its Nucleotide Sequence was 3468bp,which shared 99%homology of other reported cry9Ea in GeneBank.This sequence was submitted to the Genebank(Accession numbers was EU760456),and was designated as cry9Ea4 by the Bt cry gene Nomenclature Committee5 the vip3 gene of these 132 Bt isolates was identified.The results showed 25 isolates harbored vip3A gene and 4 isolates harbored novel vip3 genes.6 partial sequence of vip3A gene had been cloned.7 Strain ZG5-4 expressed proteins with toxicity to Aedes albopictus and pieris rapae Linne,the LC₅₀ was 23.42 ng/mL and 13.24ng/mL,respectively.Strain HY3-2 which carry vip3A gene had toxicity towards Aedes albopictus,pieris rapae Linne,Aedes with an LC₅₀ of 21.05 ng/mL,14.53 ng/mL and 34.58 ng/mL.8 The Species identification of strain ZG5-4 and showed that strain ZG5-4 was identified as Bacillus thingiensis vat galleriaea and strain HY3-2 was identified as Bacillus thringiensis vat entomocidus...