拟南芥细胞周期基因AtCDC5的功能研究及抗体制备  

Functional research and antibody preparation of Arabidopsis thaliana cell cycle gene AtCDC5

作　　者：徐金龙 梁爽[1] 郁飞[1] XU Jin-long;LIANG Shuang;YU Fei(College of Life Sciences, Northwest A&F University, Yangling 712100, China)

机构地区：[1]西北农林科技大学生命科学学院,陕西杨陵712100

出　　处：《江苏农业学报》2019年第1期26-32,共7页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(31741010)

摘　　要：MYB相关CDC5 (Cell division cycle 5)蛋白对于细胞周期G2期的正常进行是必须的,但是在植物中对这种蛋白质的研究还较少涉及。本试验测量比较野生型拟南芥(Col-0生态型)和At CDC5 T-DNA插入缺失突变体(GK_278B09)的主根生长速度和长度,利用瞬时表达At CDC5-GFP研究At CDC5在拟南芥叶片叶肉细胞中的定位,同时利用At CDC5 N端144个氨基酸在大肠杆菌中进行原核表达和纯化,免疫家兔获得针对At CDC5的多克隆特异性抗体,从表型、细胞和蛋白质水平研究At CDC5基因的功能。结果表明,GK_278B09的主根因发育受到抑制而生长速度缓慢; At CDC5-GFP融合蛋白主要定位于细胞核中;经过分离得到的At CDC5蛋白抗血清能够有效地检测出10 ng原核表达的At CDC51-144抗原,并在原生质体At CDC5过表达体系中检测出1条分子量约为120 000的条带,此条带与At CDC5-GFP大小相近。本研究成功制备出At CDC5蛋白的多克隆抗体,为进一步研究At CDC5蛋白影响细胞周期从而影响植物生长发育提供了研究基础。MYB related protein cell division cycle 5(CDC5)is essential for the G2 phase of cell cycle,but the research about CDC5 in plant is few.In the present study,the main root length and growth rate of wild-type Arabidopsis thaliana(Col-0 ecotype)and AtCDC5 T-DNA insertion deletion mutant(GK_278B09)were compared,and the subcellular localization of the AtCDC5 protein was studied by transient expressed AtCDC5-GFP.Moreover,prokaryotic expression and were applied using 144 amino acids at the N-terminus of AtCDC5 in Escherichia coli.The polyclonal antibody against AtCDC5 was obtained by immunizing rabbits.The function of AtCDC5 was researched at levels of phenotype,cell and protein.The results showed that the main root development of GK_278B09 was inhibited so that the rate of growth was lower than Col.The AtCDC5-GFP fusion protein was located in the nuclei.The separated serum of AtCDC5 could test 10 ng of AtCDC5 antigen,and a band with themolecular weight of 120 000 was detected in the protoplast system.Furthermore,the size of the band was similar to that of AtCDC5-GFP.The polyclonal antibody against AtCDC5 provides resource for the research on the function of AtCDC5 in plant growth.

关 键 词：细胞周期基因CDC5 亚细胞定位 原核表达 抗体制备 

分 类 号：Q786[生物学—分子生物学]