利用酵母双杂交系统筛选绒毛白蜡FvCAMTA1互作蛋白  

Screening of FvCAMTA1-interactive proteins in Fraxinus velutina using yeast two hybrid system

作　　者：燕丽萍[1] 吴德军[1] 王因花[1] 李丽[1] 任飞[1] 高铖铖 YAN Liping;WU Dejun;WANG Yinhua;LI Li;REN Fei;GAO Chengcheng(Shandong Provincial Key Laboretory of Forest Tree Genetic Improvement,Shandong Provincial Academy of Forestry,Jinan 250014,Shandong,China;College of Forestry,Shandong Agricultural University,Taian 271000,Shandong,China)

机构地区：[1]山东省林业科学研究院山东省林木遗传改良重点实验室,山东济南250014 [2]山东农业大学林学院,山东泰安271000

出　　处：《中南林业科技大学学报》2021年第12期111-120,共10页Journal of Central South University of Forestry & Technology

基　　金：山东省重点研发计划项目(2019GNC106143);山东省农业良种工程项目(2019LZGC009);林业知识产权转化运用项目“白蜡新品种繁育专利技术产业化”(14GNC111006)。

摘　　要：【目的】绒毛白蜡是重要的盐碱地造林树种,挖掘绒毛白蜡的耐盐潜力,对推动黄河流域盐碱地生态保护和修复具有重要意义。CAMTA是植物抗逆胁迫信号通路中的重要转录激活因子,但绒毛白蜡盐胁迫信号途径中包括FvCAMTA1在内的大部分关键基因,尚未被克隆与鉴定,与FvCAMTA1发生互作的蛋白也不清楚,严重阻碍绒毛白蜡抗盐分子机理研究的瓶颈。本研究挖掘绒毛白蜡钙调素结合转录因子FvCAMTA1的互作蛋白,为解析其抗盐性机制奠定基础。【方法】采用酵母双杂交技术对其互作蛋白进行探究,提取绒毛白蜡‘盐蜡’新品种幼苗的总RNA,利用SMART技术合成ds cDNA,依据FvCAMTA1基因的CDS序列设计引物,通过PCR扩增出FvCAMTA1基因的编码序列(约2700 bp),构建重组诱饵载体pGBKT7-FvCAMTA1转化酵母菌株Y2HGold,并在缺陷型培养基上检测诱饵载体的毒性与自激活活性,筛选与绒毛白蜡钙调素结合转录因子FvCAMTA1互作的猎物蛋白,对部分互作蛋白进行了回补验证,并对互作的猎物蛋白进行Gene Ontology(GO)注释分析。【结果】成功构建了受盐诱导的绒毛白蜡全株幼苗的cDNA酵母双杂交文库,文库滴度为4.58×10^(9)CFU·mL^(-1),插入片段长度为250~2000 bp,重组率为100%。经检测诱饵载体无法自我激活也无毒性,所筛选的猎物蛋白经测序和Blast比对分析,并对其进行了共转验证,分离得到相互作用的46个阳性克隆。GO注释显示互作蛋白参与生物过程有11种,分子功能7种,细胞组分12种。研究结果补充和完善了FvCAMTA1的信号传递途径,为进一步研究绒毛白蜡钙调素结合转录因子FvCAMTA1及其共生互作蛋白的作用机制提供了新的分子证据。【结论】成功构建了绒毛白蜡酵母双杂交文库,为鉴定FvCAMTA1的互作蛋白并解析抗盐机理研究提供了重要理论依据。【Objective】Fraxinus velutina is an important afforestation species on saline alkali land.It is of great significance to explore the salt tolerance potential of Fraxinus velutina for the promotion in ecological protection and restoration of saline alkali land in the Yellow River Basin.CAMTA is an important transcription activator in stress-resistance signal pathways of plant.However,most of the key genes,including FvCAMTA1,in salt signaling pathway of Fraxinus velutina have not been identified,and their interacting proteins are not clear,which seriously hinders the research on the molecular mechanism of salt resistance.In this study,we identified the interaction proteins of FvCAMTA1,and laid the foundation for the analysis of salt-resistance mechanism.【Method】The interacting proteins were preliminarily explored by yeast double hybrid.Total RNA was extracted from seedlings of Fraxinus velutina cultivar‘Yanla’and ds cDNA was synthesized by SMART reverse transcription.Primers were designed according to CDS sequence of FvCAMTA1 gene to construct recombinant bait vector pGBKT7-FvCAMTA1which was transformed into yeast strain Y2HGold.The cytotoxicity and self-activation effect of the bait were detected.The prey protein interacting with the FvCAMTA1 was screened and were analyzed by Gene Ontology(GO).【Result】The cDNA library of salt-induced ash was used in yeast hybrid,with titer of 4.58×10^(9)CFU·mL^(-1),length of inserted fragment from 250 to 2000 bp,and 100%of recombination efficiency.There was no cytotoxicity or self-activation effect.The results of sequencing and Blast analysis showed 46 obtained interacting proteins,including 11 proteins in biological processes,12 for cell components and 7 for molecular functions.The results improve our understanding of the signal transduction of FvCAMTA1,and provide new molecular evidence for further study on the symbiotic interaction mechanism.【Conclusion】In this study,a yeast two hybrid library of Fraxinus velutina was constructed to identify the interaction

关 键 词：绒毛白蜡 FvCAMTA1 酵母双杂交 蛋白互作 GO分析 

分 类 号：S718.46[农业科学—林学]