橡胶树棒孢霉落叶病菌酵母单杂交cDNA文库及CcCas5启动子诱饵载体的构建

Construction of a yeast one-hybrid cDNA library and CcCas5 promoter bait vector of Corynespora cassiicola causing leaf fall disease of Hevea brasiliensis

为了筛选出与CcCas5启动子结合的转录因子,本研究构建了橡胶树棒孢霉落叶病菌的酵母单杂交cDNA表达文库和CcCas5启动子诱饵载体,并对3-氨基-1,2,4-三唑(3-amino-1,2,4-triazole,3-AT)的最佳抑制浓度进行了筛选。结果显示,构建的酵母单杂交cDNA表达文库总容量为3.49×10^(6)cfu,插入片段主要分布于750~2000 bp之间,重组率为95.8%;克隆了CcCas 5基因2600 bp的启动子序列,预测获得真菌主要的10大类转录因子的结合位点165个;构建了分别携带1500 bp和1000 bp启动子序列的酵母单杂交启动子诱饵载体,10 mmol/L的3-AT能抑制pHis2.1-pCcCas5-1000和pHis2.1-pCcCas5-1500的背景表达。试验结果为通过酵母单杂交筛选与CcCas5启动子结合的转录因子和进一步研究转录因子对CcCas5表达的调控作用奠定基础。

In order to screen the transcription factors that bind to the CcCas5 promoter,a yeast one-hybrid cDNA expression library of Corynespora cassiicola and a CcCas5 promoter bait vector were constructed,and the optimal inhibitory concentration of 3-amino-1,2,4-triazole(3-AT)was screened in this study.The results showed that the total capacity of the constructed yeast one-hybrid cDNA expression library was 3.49×10^(6)cfu,the inserted fragments mainly distributed between 750 to 2000 bp,and the recombination rate was 95.8%.The 2600 bp promoter sequence of CcCas5 was cloned and the binding sites of 165 main fungal transcription factors in 10 families were predicted.Yeast one-hybrid promoter bait vectors carrying 1500 bp and 1000 bp promoter sequences were constructed,respectively,and 10 mmol/L of 3-AT could inhibit the background expression of pHis2.1-pCcCas5-1000 and pHis2.1-pCcCas5-1500.The above results lay the foundation for screening transcription factors that bind to the CcCas5 promoter through yeast one-hybrid screening and further studying the regulatory effects of transcription factors on CcCas5 expression.