番茄分裂泛素酵母双杂膜系统cDNA文库及SlToc159诱饵载体的构建  

Construction of tomato cDNA library and SlToc159 bait vector based on split ubiquitin system

作　　者：王琪[1,2] 闫见敏 岳江[1,2] 李云洲 须文 WANG Qi;YAN Jianmin;YUE Jiang;LI Yunzhou;XU Wen(College of Agriculture,Guizhou University,Guiyang 550025,China;Vegetable Research Institute,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学农学院,贵阳550025 [2]贵州大学蔬菜研究院,贵阳550025

出　　处：《植物生理学报》2022年第5期825-834,共10页Plant Physiology Journal

基　　金：国家自然科学基金(31960604);贵州省省级科技计划项目(黔科合基础[2019]1107号、黔科合基础-ZK[2021]重点040);贵州大学人才引进科研项目(贵大人基合字[2014]18号);贵州省生物学国内一流学科建设学科开放基金(GNYL[2017]009FX4KT43)。

摘　　要：叶绿体是植物进行光合作用、脂质代谢以及氨基酸合成的重要场所。叶绿体自身基因组编码的蛋白质仅有100个左右,其余95%(约3000多个)的叶绿体蛋白质即前体蛋白是由细胞核基因组编码的,在细胞质中翻译后通过TOC-TIC转运蛋白复合体导入叶绿体的特定位置而发挥生物学功能。Toc159是叶绿体外膜转运蛋白,主要负责转运初始阶段前体蛋白的特异性识别。为研究番茄SlToc159与前体蛋白的互作机制,本试验以‘Alisa Craig’番茄为材料,提取番茄幼苗叶片总RNA,采用Gateway技术构建分裂泛素化膜系统cDNA文库,同时构建诱饵载体pBT3-N-Toc159AGM和pBT3-N-Toc159GM,并用营养缺陷法检测其自激活活性。文库质量检测结果显示,文库总容量为1.6×10^(7)CFU,文库滴度为4.0×10^(6)CFU·mL^(-1),外源基因插入片段为750~2000 bp,重组率为100%,符合后续筛选互作蛋白的cDNA文库要求。酶切及测序结果表明,诱饵载体pBT3-N-Toc159AGM和短截A区的pBT3-N-Toc159GM构建成功,能够在分裂泛素化系统中正确表达且无自激活活性。本研究结果为番茄膜蛋白利用酵母双杂交筛选互作蛋白提供参考与理论基础。Chloroplasts are important sites for photosynthesis,lipid metabolism and amino acid synthesis in plants.The chloroplast’s own genome codes for only about 100 proteins,and the remaining 95%of chloroplast proteins(more than 3000)are encoded by nuclear genome and translated in the cytoplasm,and then imported into the specific locations of chloroplast through TOC-TIC complex to perform biological functions.Toc159,a chloroplast outer membrane transporter,is responsible for the specific recognition of precursor proteins at the initial stage of transport.In order to study the interaction mechanism between Sl Toc159 and precursor protein in tomato,total RNA was extracted from tomato seedling leaves using’Alisa Craig’as material,and cDNA library of split ubiquitin membrane system was constructed by Gateway technique.Meanwhile,bait vectors of pBT3-N-Toc159AGM and pBT3-N-Toc159GM were constructed,respectively.Bait vectors’self-activation activity was detected by nutritional deficiency assay.cDNA library quality test results showed that the total volume of the library was 1.6×10^(7) CFU,the titer of the library was 4.0×10^(6) CFU·mL^(-1),the inserted fragment of exogenous gene was 750-2000 bp,and the recombination rate was 100%,which met the requirements of cDNA library screening for interacting proteins.The results of enzyme digestion and sequencing showed that both of bait vector pBT3-N-Toc159AGM and pBT3-NToc159GM in truncated A domain were successfully constructed and could be expressed correctly in split ubiquitination system without self-activation activity.The results of this study provide reference and theoretical basis for screening interaction proteins of membrane proteins using yeast two-hybrid in tomato.

关 键 词：番茄 分裂泛素酵母双杂交系统 CDNA文库 GATEWAY技术 叶绿体外膜转运蛋白 SlToc159 诱饵载体 

分 类 号：S641.2[农业科学—蔬菜学]