麦根腐平脐蠕孢cDNA文库的构建及BsTup1互作蛋白筛选

Construction of cDNA library of Bipolaris sorokiniana and screening of BsTup1 interacting proteins

【目的】深入探究麦根腐平脐蠕孢(Bipolaris sorokiniana)生长发育及致病力的分子作用机制,并鉴定BsTup1的互作蛋白。【方法】利用麦根腐平脐蠕孢(B.sorokiniana)孢子和不同时期的菌丝体为材料,构建酵母双杂交cDNA文库,以BsTup1基因为诱饵来筛选酵母双杂交文库,确定与BsTup1相互作用的蛋白。【结果】1)利用SMART(switching mechanism at 5′end of the RNA transcript)技术首次成功构建了麦根腐平脐蠕孢(B.sorokini-ana)分生孢子和菌丝体的混合cDNA文库。文库鉴定结果表明,构建的cDNA文库库容为4.8×10^(7) cfu·mL^(-1),文库插入片段重组率达100%且平均大小为1000 bp。2)构建了pGBKT7-BsTup1诱饵载体,无自激活活性。3)使用诱饵蛋白载体pGBKT7-BsTup1对麦根腐平脐蠕孢(B.sorokiniana)酵母双杂交cDNA文库进行筛选,经测序、序列比对和酵母回转验证,获得38个与BsTup1相互作用的候选蛋白。【结论】成功构建了麦根腐平脐蠕孢(B.sorokiniana)的cDNA文库,并鉴定出38个与BsTup1相互作用的候选蛋白。

【Objective】To investigate the molecular mechanisms of growth,development,and pathogenicity of Bipolaris sorokiniana and identify the interacting proteins of BsTup1.【Method】We used spores and mycelia at different stages of B.sorokiniana as materials to construct a yeast two-hybrid cDNA library,and screened the yeast two-hybrid library by BsTup1 gene as the bait for determining the proteins interacting with BsTup1.【Result】1)The mixed full-length cDNA library of conidia and mycelium was successfully constructed for the first time using SMART(switching mechanism at 5′end of the RNA transcript)technology.The results of library identification showed that the constructed cDNA library had a capacity of 4.8×10^(7) cfu·mL^(-1),the library insertion fragment recombination rate reached 100% and the average size was around 1000 bp.2)The bait protein vector pGBKT7-BsTup1,a transcriptional inhibitor of BsTup1 in B.sorokiniana,successfully constructed and confirmed no self-activation activity.3)Using this bait vector,38 candidate proteins interacting with BsTup1 were obtained after sequencing,sequence alignment,and yeast verification.【Conclusion】We successfully constructed a cDNA library of B.sorokiniana,and identified 38 candidate proteins which interacted with BsTup1.