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
胡国豪 1杨子平 2刘铜 1张荣意 1侯巨梅1
作者信息
- 1. 海南大学热带农林学院,海南省农用生物制剂创制工程研究中心,海口 570228
- 2. 中国热带农业科学院南亚热带作物研究所,湛江市热带作物遗传改良重点实验室,湛江 524091;中国热带农业科学院三亚研究院,三亚 572025
- 折叠
摘要
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3.49×106 cfu,插入片段主要分布于750～2 000 bp之间,重组率为95.8％;克隆了 CcCas5基因2 600 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1 500 bp和1 000 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.
Abstract
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 × 106 cfu,the inserted fragments mainly distributed between 750 to 2 000 bp,and the recombination rate was 95.8％.The 2 600 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 1 500 bp and 1 000 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.
关键词
橡胶树/多主棒孢/CcCas5/启动子/酵母单杂交/cDNA文库Key words
Hevea brasiliensis/Corynespora cassiicola/CcCas5/promoter/yeast one-hybrid/cDNA library引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
万方数据