基于家族基因分析的甘蓝MLPK互作PUB蛋白的筛选

Screening of Brassica oleracea MLPK-interacting PUB Proteins Based on Gene Family Analysis

MLPK(M–位点受体激酶)是芸薹属自交不亲和正向调控关键元件,其参与自交不亲和信号传导的分子机制尚不明确,同时自交不亲和下游信号元件也有待于进一步分离。为了探索分离MLPK互作蛋白的思路和方法,构建了不含核定位信号的MLPK短截蛋白(MLPK-T),并利用酵母双杂交检测到MLPK与臂重复蛋白1(ARC1)作用,通过全基因组鉴定分别获得了96个甘蓝、101个白菜、70个琴叶拟南芥和62个拟南芥PUB蛋白,其中含有臂重复序列的PUB蛋白共为127个。通过系统进化分析,筛选到8个含臂重复序列的甘蓝BoPUB蛋白,其8个基因全部在柱头内表达,且成功利用酵母双杂交检测到MLPK与3个含臂重复序列的BoPUB蛋白Bol008579、Bol016165和Bol023511相互作用。

MLPK(M-locus protein kinase)is a positive key mediator of Brassica self-incompatibility signaling,however,the molecular mechanism of MLPK in mediating Brassica self-incompatibility response is still unknown.Meanwhile,the downstream factors of Brassica self-incompatibility signaling still need to be isolated.Aim to explore the idea and methods to isolate MLPK-interacting proteins,here,the MLPK truncated protein MLPK-T lack of the plasma membrane localization motif was constructed,the interaction of MLPK with ARC1 was proved via conventional yeast two-hybrid system.Then,we obtained 96,101,70 and 62 PUB proteins from B.olercea,B.rapa,Arabidopsis lyrata and A.thaliana through genome-wide identification,amount to 127 PUB proteins containing ARM repeat domains.Based on phylogenetic analysis,we selected 8 PUB proteins,and RT-PCR showed that all these 8 genes expressed in B.oleracea stigmas.Finally,the interaction of MLPK with Bol008579,Bol016165 and Bol023511 were proved through yeast two-hybrid system.