辣椒胎座cDNA酵母文库构建和鉴定  

Construction and Identification of Yeast cDNA Library of Chili Pepper(Capsicum annuum)Placenta

作　　者：黄祝兵 朱张生[1] 张霜霖 宋佳丽[1] 陈长明[1] 雷建军[1] Huang Zhubing;Zhu Zhangsheng;Zhang Shaunglin;Song Jiali;Chen Changming;Lei Jianjun(Key Laboratory of Horticultural Crop Biology and Germplasm Innovation in South China,Minstry of Agriculture,Collge of Horticulture,South Chi-na Agricultural University,Guangdong,510642)

机构地区：[1]华南农业大学园艺学院,农业部热带亚热带种质资源创制和利用重点实验室,广州510642

出　　处：《分子植物育种》2020年第13期4317-4323,共7页Molecular Plant Breeding

基　　金：国家自然科学基金项目(C150203)资助。

摘　　要：辣椒素类物质是辣椒属植物特有的一种次生代谢产物,特异地在辣椒果实胎座中合成,且具有明显的发育阶段特异性。辣椒素合成途径相关基因主要在辣椒花后16 d胎座发育至绿熟期转录,然而转录调控的精细分子机制尚不太清楚。用已知参与调控辣椒素生物合成的转录因子为诱饵,采用酵母双杂交技术从辣椒素合成阶段酵母cDNA文库中筛选与其互作的蛋白,有利于明确辣椒素生物合成的转录调控机制。因此,获得辣椒素合成阶段的高质量酵母cDNA文库是关键。本研究采用辣椒‘59’自交系花后16 d(DPA)的RNA作为模板,之后用SMART c DNA/均一化c DNA文库技术(Switching mechanism at 5’end of the RNA transcript)与同源重组的方法将‘59’自交系的胎座cDNA文库转化酵母感受态Y187。通过对收集的文库进行鉴定发现,滴度达到3.63×10~7CFU/m L,插入片段大约250~2000 bp,文库满足酵母双杂交要求。辣椒胎座高质量cDNA文库的构建为后续辣椒素生物合成的转录调控网络研究提供了基础。Capsaicinoids are secondary metabolites restricted to the genus Capsicum specific synthesized in chili pepper(Capsicum annuum)placenta and are synthesized dependent on the developmental stage of the fruits.Capsaicin synthesis pathway-related gene are mainly transcribed from 16 days post-anthesis(DPA)to green mature stage,however the molecular mechanism of transcriptional regulation is still not clear.Using transcription factor known to be involved in the regulation of capsaicin biosynthesis as bait for screening and interacting proteins from yeast cDNA using yeast two-hybrid technique is useful for clarifying the transcription regulation mechanism of capsaicin biosynthesis.High quality yeast c DNA library is key to success of this experiment.The total RNA was isolated from chili pepper(Capsicum annuum)inbred line’59’placenta in 16 days post-anthesis(DPA).By using SMART technique and homologous reorganization method,we constructed the chili pepper inbred line’59’placenta cDNA library in yeast strain Y187.The qualities of the cDNA libraries were evaluated as the followings:the capacity of cDNA library was 3.63×10~7 CFU/mL,PCR results showed that the inserts fragments size varied from250 to 2000 bp.This library can meet yeast two-hybrid requirements.The library lays the foundation for studying transcriptional regulatory network of capsaicin biosynthesis.

关 键 词：辣椒素类物质 辣椒 酵母 胎座cDNA文库 

分 类 号：S641.3[农业科学—蔬菜学]