Cloning And Function Analysis Of BbThi, A Thiazole Biosynthetic Gene In Beauveria Bassiana

Entomopathogenic fungi are a promising candidate for microbial control of many agricultural pests in nature.They invade the host directly,infect pest circularly and host can rarely develope resistance to them.Therefore,they have distinct superiority on controlling piercing and sucking type pests and have large practical potentials.However, the systematic theory of its development and pathogenicity is too limited,which hampered the further exploration and application of mycoinsecticedes.Under such circummustances,it is necessary to explore the mechanisms of pathogenesis and elucidate more related gene functions in order to create a market-oriented and environment-friendly mycoinsecticede.Beauveria bassiana is one of the most important entomopathogenic fungi and has been widely used as a biological agent for decades.A mutant M936 with conidiation-defection morphology was screened from the the T-DNA insertional mutant library of B.bassiana.Then,a thiazole biosynthetic gene BbThi was cloned from M936 through YADE(Y-shaped adaptor-dependent extension)method(GeneBank acession No.of BbThi is EF122790).Three homlogous recombination strains(△BbThi26,△BbThi33,△BbThi48)were obtained through dual-selection(positive and negative) based Agrobacterium tumefaciens-mediated transformation of B.bassiana.One heterogenous recombination transformant(T65)and one BbThi over-expressing strain (Othi)were also gained.Finally,the characteristics and function of BbThi were primarily elucidated.The whole DNA sequence of BbThi is 2720 bp,with a 1403 bp promotor and a 1100 bp gene.The pormotor contains six CAAT boxes and three STREs(stress responsive element).The open reading frame of BbThi is 993 bp,which encodes a protein of 330 amino acids with a calculated Mr of 35.25 kDa and pI of 5.89.There is an adenosine diphospho-5-(β-ethyl)-4-methylthiazole-2carboxylic acid(ADT)site in the protein,which is shared by the THI4 protein family.Phylogenetic analysis indicated that BbTHI has high similarity to thizole biosynthetic enzymes from other filamentous fungi,but relatively lower similarity to Saccharomyces cerevisiae.Southern blotting result indicated that BbThi existed in B.bassiana genome as a single copy.The defective morphology of the BbThi disrupted mutants could be restored by adding exogenous VB1 to the medium,and endogenous VB1 contents in the mutants were largely reduced compared with the wild type strain,which indicated that BbThi takes part in the VB1 biosynthesis pathway in B.bassiana.Disruption of BbThi decreased the conidia yields significantly and influenced the colony morphology of B. bassiana.However,disruption of BbThi has no obvious effect on the conidia germination and virulence of B.bassiana.