月季酵母双杂交cDNA文库的构建及RhRD22互作蛋白的筛选  

Construction of a High-quality Yeast Two-hybrid cDNA Library and Screening of the Proteins Interacting with RhRD22 of Rose

作　　者：张竹君 任文娟 耿子雯 付鲁峰 李伟 姜新强 刘庆华 Zhang Zhujun;Ren Wenjuan;Geng Ziwen;Fu Lufeng;Li Wei;Jiang Xinqiang;Liu Qinghua(College of Landscape Architecture and Forestry,Qingdao Agricultural University,Qingdao,266109)

机构地区：[1]青岛农业大学园林与林学院,青岛266109

出　　处：《分子植物育种》2021年第7期2243-2255,共13页Molecular Plant Breeding

基　　金：国家重点研发计划项目(2018YFD1000400)资助。

摘　　要：月季(Rosa hybrida)是全球重要的观赏植物,其生长发育和生产质量严重受到干旱胁迫的制约。在已有月季转录组数据的基础上,干旱胁迫可以诱导干旱响应基因RD22(dehydration-responsive gene RD22)的表达。为了进一步探究RhRD22参与干旱胁迫的分子机制,本研究利用改良的SMART(Switching Mechanism At 5’end of RNA Transcript)技术构建月季干旱胁迫酵母双杂交cDNA文库,并利用酵母双杂交技术筛选与RhRD22相互作用的未知潜在蛋白。结果表明:成功构建了高质量月季酵母双杂交cDNA文库,该文库库容为3×106cfu;插入cDNA片段的长度分布在400~2000 bp,平均长度大于1000 bp;重组率约为100%。此外,利用重组诱饵蛋白p GBKT7-RhRD22在该文库中筛选了7个与RhRD22相互作用的蛋白,包括RhEF-Ts、RhDRT100、RhGAPN、RhLDH、RhACLA-3、RhCALS3、RhEOL1。互作蛋白的GO注释分析表明,RhRD22可能参与蛋白质多肽链的翻译和延伸、DNA修复、代谢反应、乙烯生物合成、蛋白质泛素化、植物防御、应激反应等生物学过程。上述结果为进一步解析RhRD22参与干旱胁迫的分子机制提供了理论参考。Rose(Rosa hybrida) is an important ornamental plant worldwide, its growth and productivity are seriously restricted by drought stress. Based on the existing transcriptomic data of Rosa hybrida, the dehydration-responsive gene RD22 could be induced by drought stress. To further explore the molecular mechanism of RhRD22 involved in drought stress, the improved SMART(Switching Mechanism At 5’end of RNA Transcript) technology was used to construct a yeast two-hybrid c DNA library under drought stress in Rosa hybrida, and the unknown potential interacting proteins of RhRD22 were screened by yeast two-hybrid technology in the study. The results showed that a high-quality yeast two-hybrid c DNA library was successfully constructed. The library capacity was 3×106 cfu, the length of the inserted cDNA fragments emphasized on 400~2 000 bp, the average length was more than 1 000 bp,and the recombination rate was about 100%. In addition, the recombinant bait protein pGBKT7-RhRD22 was used to screen seven proteins interacting with RhRD22 in the cDNA library, including RhEF-Ts, RhDRT100, RhGAPN,RhLDH, RhACLA-3, RhCALS3 and RhEOL1. The GO annotation analysis of the interacting proteins illustrated that RhRD22 may be involved in the translation and extension of protein polypeptide chains, DNA repair, metabolic reactions, ethylene biosynthesis, protein ubiquitination, plant defense, stress response and other biological processes.These results above provided a theoretical reference for further exploring the molecular mechanism of RhRD22 involved in drought stress.

关 键 词：月季(Rosa hybrida) RhRD22 c DNA文库 酵母双杂交技术 互作蛋白 

分 类 号：S685.12[农业科学—观赏园艺]