Chitinase Gene Cloning And High Virulence Genetic Engineering Strain Acqisition Of Verticillium Lecanii

Verticillium lecanii as an important worldwide entomopathogenic fungus, plays asignificant role in the biological control against pests with piercing-suckingmouthparts. However, it has some inevitable disadvantages such as a longer period ofkilling pests and higher requirements of temperature and humidity, which haveseriously restricted its application. Genetic improvement of V. lecanii could solvethese problem. Research on pathogenicity of entomopathogenic fungus indicated thatchitinase may be a important toxic factor in the stage of infesting pest, because itcould hydrolyze insect cuticle in collaboration with proteases.Research rencentlyindicates that overexpressing chitinase was shown to enhance virulence of other kindsof entomopathogenic fungus.However, little is researched on V. lecanii. In this paper,three endochitinase genes from three strains of V. lecanii were cloned, and theirnucleic acid sequences and amino acid sequences were analyzed with bioinformaticmethods. Also high virulence strain with overexpression chitinase was obtained bygenetic modification.Three full length cDNA sequences which1567bp,1581bp and1562bp lengthrespectively were produced by RACE PCR (Genbank accession number: JN969371,JN982325and JN982326). Homology of the sequences was up to91.84%. The genesthat contained a open reading frame (ORF) could encode a proteins of427,429and429amino respectively. The sequence analysis of deduced amino acid revealed thatthe predicted molecular weight of precursor protein were46.5kDa,46.7kDa and46.9kDa respcetively and these proteins were considered to be one kind of stable,hydrophilic acid protein. Tertiary structure analysis showed all three precursor proteinhave a α/β structure. α-helices were located in the outer and β-sheet wrapped in themedial. Structure of all three proteins are closely, roughly spherical.The ORF of ChitVl6063,ChitV3450and ChitVp28were inserted into the fungalexpression vector pBARGPE-1which contain strong promoter and terminatorrespectively to construct a chitinase overpressing plasmid.Then transformed thewild-type strain with blastospore transformation method. The results of bioassayrevealed that virulence of the three transgenic strains on the toxoptera aurantii wereconsiderably much higher than that of wild-type strain. Values of LC50were reducedto3.43%,1.72%and1.23%, respectively and Values of LT50was shortened by29.51%,30.46%and33.90%, which indicated that the strain with overexpressedtransgenic chitinase could significantly improve the virulence of transformants.