棉花GhMADS43基因的克隆及酵母自激活分析  

Cloning and Yeast Self-activated Detection Analysis of GhMADS43 Gene in Cotton

作　　者：张晓红 李晓琪 张峥 胡根海[1] ZHANG Xiaohong;LI Xiaoqi;ZHANG Zheng;HU Genhai(College of Life Science and Technology,Henan Institute of Science and Technology,Henan Collaborative Innovation Center of Modern Biological Breeding,Xinxiang 453003,China)

机构地区：[1]河南科技学院生命科技学院,现代生物育种协同创新中心,河南新乡453003

出　　处：《华北农学报》2020年第6期15-21,共7页Acta Agriculturae Boreali-Sinica

基　　金：棉花生物学国家重点实验室开放课题(CB2018A08);转基因生物新品种培育科技重大专项(2020ZX08009-12B)。

摘　　要：为探索棉花GhMADS43基因在茎尖和花分生组织发育过程中的机理,从棉花中克隆了MADS-box家族中GhMADS43基因,并对该基因进行表达分析和酵母自激活分析,以期利用酵母双杂交筛选GhMADS43的互作蛋白质。对棉花中GhMADS43基因进行克隆及组织特异性表达分析,结果显示,GhMADS43基因全长696 bp,编码231个氨基酸,与其他植物中编码FUL蛋白的基因亲缘关系相近。由特异性表达分析可得,GhMADS43基因在根和顶芽中表达量较高;对2种不同果枝材料进行不同顶芽发育时期的表达分析表明,该基因在无限果枝材料中棉所50五叶期和六叶期的表达量显著高于一式果枝材料早铃1号。将该基因构建到酵母诱饵载体pGBKT7,得到pGBKT7-GhMADS43诱饵表达载体。重组质粒转化酵母Y2HGold菌株后,在SD/-Trp平板上生长良好,表明重组诱饵质粒表达产物对酵母细胞无毒性,在SD/-Trp/X-α-Gal平板上长出白色菌落变蓝。利用不同浓度的3-AT对其进行筛选发现,该基因不能在三缺培养基SD/-Trp/-His/-Ade中生长,表明该基因不具有自激活活性。构建好诱饵表达载体后,可进一步从棉花酵母双杂交cDNA文库中筛选GhMADS43的互作蛋白质,为将来研究该基因在茎尖和花分生组织发育过程中的功能和调控通路提供基础。In order to elucidate the cotton GhMADS43 gene in shoot apical meristem and floral meristem development and to screen for proteins that interact with this geneby yeast two hybrid system.This study cloned the coding sequences of GhMADS43 and analyzed the expression of this gene in different tissues and shoot apical meristem periods.The results showed that the GhMADS43 gene was 696 bp in length and encoded 231 amino acids,which was similar to FUL in other plant species.According to specific expression analysis,GhMADS43 gene had higher expression in roots and apical buds,and the expression of the gene was higher in infinite fruit branches cotton CCRI50 than that of one fruit branches cotton Zaoling No.1.We also constructed bait vectorsp pGBKT7-GhMADS43 and transformed it into the yeast strain Y2 HGold.The results showed that all the Y2 HGold strains containing the recombinant plasmid grew well on the SD/-Trp media.It suggested that the recombinant bait proteins were no toxic to yeast.The transformed yeast strains developed blue plaques on the SD/-Trp/X-α-Gal media.And they could not grow on the SD/-Trp/-Ade/-His media with different concentration of 3-AT.The results indicated that the GhMADS43 gene had no self-activating activity.The constructed bait vector can be used for further screening of proteins interacting with GhMADS43 from yeast two-hybrid cDNA library in cotton.The study plays the foundation for analyzing of the gene function and regulatory pathways in shoot apical meristem and floral meristem development in the future.

关 键 词：棉花 分生组织发育 GhMADS43 表达分析 自激活 

分 类 号：Q78[生物学—分子生物学] S562.03[农业科学—作物学]