Cloning And Functional Analysis Of The Herbicidal-Activity-Related Gene In Botrytis Cinerea

The Botrytis cinerea wild type BC4 and some mutants(the high herbicidal active strains and low herbicidal active strains)have been analyzed by DDRT-PCR in our laboratory,a 694 bp gene fragment which is possibly a herbicidal active related gene have been obtained.In this study,the 694 bp gene fragment was extended to 3961 bp by Genomic Walking using two steps of 5'Genomic Walking and two steps of 3'Genomic Walking.The 3961 bp gene was analyzed by the software of BLAST on the website ncbi,it is indicated that the homology between CaMK gene of Botryotinia fuckeliana and the cloned gene of BC4 is 98%.According to the GenBank reported Botryotinia fuckeliana whole CDS,we cloned the cDNA of BC4 CaMK gene by RT-PCR.The analysis of Southern blotting indicated that there was a single copy of CaMK gene in the genomic.The bioinformatics of CaMK gene have been analyzed.The CaMK cDNA and DNA was compared by DNAMAN,it is indicated that the CaMK gene contains 7 extrons and 6 introns.The CaMK protein character was predicted by DNAStar,it is indicated that the predicted protein molecular is 81.8748 kD,the protein has 730 amino acids which containing 95 alkaline amino acids(K,R),116 acidic amino acids(D,E),216 hydrophobous amino acids(AILFWV),160 polar amino acids(NCQSTY),the isoelectric point is 6.22.The conserved domain have been analyzed by ScanPro site,it is indicated that the primary structure of CaMK gene contains ATP-binding region signature and Serine/Threonine protein kinases active-site signature.The secondary structure of CaMK protein has been analyzed by SOPMA,and the three-dimensional mode has been constructed by SWISS-MODEL.The probable promoter sequences upstream the ORF 1330 bp have been analyzed by softberry.The recombinant CaMK plasmid was constructed,and it was successefully expressed in E.coli BL21.The result indicated that the expressed recombinant protein is in praecipitatum.This study is helpful to purify the protein and prepare polyclonal antibody in order to research further function of the gene.The conidium of Botrytis cinerea have been treated with CaMK inhibition KN-62, The result indicated that the inhibition rate of 60μM KN-62 to conidial germination is 50%and the inhibition rate of 80μM KN-62 to conidial germination is more than 98%;the pathogenicity of Botrytis cinerea treated with 80μM KN-62 remarkably reduced,the herbicidal activity of Botrytis cinerea treated with 80μM KN-62 remarkably weakened. It is indicated that CaMK play an important regulate role in the conidial germination, pathogenicity and herbicidal activitive substances of Botrytis cinerea.