蜡梅CpBEAT3启动子克隆及其互作蛋白的初步验证

Cloning of CpBEAT3 Gene Promoter from Chimonanthus praecox and Preliminary Verification of Its Interacting Protein

苯甲醇乙酰基转移酶(benzyl alcohol acetyltransferase,BEAT)能够以苯甲醇和乙酰辅酶A(acetyl-CoA)为底物催化合成乙酸苄酯,是植物乙酸苄酯生物合成代谢途径下游的关键酶。CpBEAT3是蜡梅叶中调控乙酸苄酯生物合成的关键基因,为了解析其在蜡梅乙酸苄酯生物合成中的转录调控机制,本研究克隆得到了CpBEAT3基因启动子区序列,使用生物信息学软件预测分析了该基因核心启动子区、顺式作用元件、CpG岛等结构特征,并通过酵母单杂交技术筛选可能与CpBEAT3结合的上游调控因子。结果表明,2115 bp的CpBEAT3核心启动子区可能位于-1844~-1794 bp处,有一个位于-1869~-1459 bp处且长度为410 bp的CpG岛,含有多种与光响应、激素调节、胁迫响应相关的顺式作用元件及多个转录因子结合位点。通过酵母单杂交技术筛选获得1个MYB类的转录因子,可以与CpBEAT3启动子结合。

Benzyl alcohol acetyltransferase(BEAT)can catalyze the synthesis of benzyl acetate using benzyl alcohol and acetyl CoA as substrates,and is a key downstream enzyme of the plant benzyl acetate bio-synthesis pathway.CpBEAT3 is a key gene regulating the biosynthesis of benzyl acetate in leaves of Chimonan-thus praecox.In order to elucidate the transcriptional regulatory mechanisms of CpBEAT3 in biosynthesis of benzyl acetate in C.praecox,the promoter region sequence of CpBEAT3 was cloned in this study.Its structural features such as core promoter region,cis-acting elements and CpG islands were predicted and analyzed using bioinformatics softwares and the upstream regulatory factors which might bind to CpBEAT3 gene were screened through yeast one-hybrid technology.The results indicated that the 2115 bp of CpBEAT3 gene core promoter region might be located at-1844~1794 bp,which had a CpG island with the length of 410 bp located be-tween-1869 and-1459 bp,and contained various cis-acting elements related to light response,hormone regulation and stress,as well as multiple transcription factor binding sites.One MYB class of transcription fac-tor was obtained through yeast one-hybrid technology,which could bind to the promoter of CpBEAT3.