Cloning Of Chitinase And Endoglucanase Gene From Bacillus Spp. And Construction Of Recombinant Bacillus Isolates

This article dealt with cloning and sequencing of chitinase and endoglucanase genes of Bacillus spp. and recombinant biocontrol isolates of Bacillus spp. by transformation with the genes.Plant disease biocontrol bacteria Bacillus subtil is ApllS and B. megaterium Ap25 were isolated from wheat field soils collected from South Australia and Tai an. Enzyme activity analysis on chitin agar and ABP media showed that B. subtilis ApllS secreted chitinase and B. megaterium Ap25 secreted endoglucanase, respectively.PCR methodology was adopted for cloning of chitinase encoding genes. Based on the sequences of chitinase gene from GeneBank, the primers for chitinase gene amplification were designed. PCR fragment was ligated with pMD18~T vector and transformed into E. coli DH5 a . Four colonies of transformed E. coli DH5 a with clear hydroiyzing zone on the chitin agar were obtained. The gene fragment in these isolates was identified by the methods of plasmid processing. DNA sequencing analysis showed that sequence homology between PCR fragment and chromosomal DNA of B. subtilis from 2599451 to 2812870 was 85%, and was 30% between the fragment and the genes encoding for chitinase of Bacillus (including B. subtilis) in GeneBank. It was thought to be a new gene encoding for chitinanse of B. subtilis.Genome library methodology was adopted for cloning of endoglucanase encoding genes. Genoraic DNA of B. megaterium was partially digested by restriction enzyme Sau3A and was used to establish the genomic library in plasmid pBluscripts. Selection for endoglucanase positive clones from transformed E. coli was carried out on the CMC medium. Seventy-four clones that showed hydrolysis ability on the CMC plate were obtained. Sequencing analysis showedthat the sequence of endoglucanase fragment exhibits 35% homology with B. subtil is chromosomal DNA(from glyB to aprE), and 27% homology with Bacillus sp. BP23 celB genes, B.pwnilus endoglucanase and B. polymyxa beta-1, 4-endoglucanase 'genes, respectively. It was recognized as a new gene encoding for endoglucanase of B. mega terium.The recombinant plasmid TvCHI (pMD18~T inserted with chitinase encoding gene from ApllS) and E. coli-Bacillus shuttle vector pHYSOOPLK were digested by EcoRI and Sail completely, and the chitinase gene was ligated with shuttle vector, and the recombinant vector was used to transform B. megaterium Ap25 competent cell. PCR and enzyme acitivity check on chitin agar showed that the chitinase gene fragment existed and expressed in the wildtype strains. Antagonistic activity test in vitro suggest that the transformants remained the ability to produce antibiotics. The recombinant strains showed an increased biocontrol efficacy against wheat take-all and Rhizoctonia sheath blight in greenhouse.