甘蓝自交不亲和相关基因BoPUB9的克隆及表达分析  

Molecular cloning and expression analysis of BoPUB9 in self-incompatibility Brassica oleracea

作　　者：谢琴琴 左同鸿 胡燈科 刘倩莹 张以忠 张贺翠[1] 曾文艺 袁崇墨 朱利泉[1] XIE Qin-Qin;ZUO Tong-Hong;HU Deng-Ke;LIU Qian-Ying;ZHANG Yi-Zhong;ZHANG He-Cui;ZENG Wen-Yi;YUAN Chong-Mo;ZHU Li-Quan(College of Agronomy and Biotechnology,Southwest University,Chongqing 400716,China)

机构地区：[1]西南大学农学与生物科技学院,重庆400716

出　　处：《作物学报》2022年第1期108-120,共13页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31572127);中央高校基本科研业务费专项资金(XDJK2020D024)资助。

摘　　要：自交不亲和性(self-incompatibility,SI)是指植株的雌蕊对自身花粉和异体花粉进行识别从而抑制自身花粉萌发的特性,PUB(Plant U-Box)蛋白在植物抗逆及信号转导过程中起着重要的作用。本研究通过分析甘蓝自花授粉0~60 min的柱头转录组数据,筛选到1个受自花授粉诱导上调表达的PUB蛋白编码基因BoPUB9。BoPUB9 cDNA序列长度为1368 bp,gDNA序列全长1720 bp,含有1个臂重复结构域和1个U-box结构域。荧光定量PCR结果显示,BoPUB9基因在甘蓝的不同组织中均有表达,在萼片中的表达量最高,花瓣、雄蕊、柱头表达量次之,在花粉花蕾中表达量相对较低,同GUS染色结果保持一致。BoPUB9基因在自花授粉0~30 min内快速上调表达,15 min后为异花授粉后表达量的20多倍,在30 min后达到差异最大,自花授粉后的表达量为异花授粉的40多倍。亚细胞定位结果显示,BoPUB9蛋白在细胞核和细胞质中均有表达,且BoPUB9蛋白在大肠杆菌中被成功诱导表达,相对分子质量为51 kD,与预测结果一致。酵母双杂交与poll-down结果显示,SRK胞内域与PUB9蛋白存在相互作用。推测BoPUB9可能是一种参与甘蓝自交不亲和反应过程的新基因,这为甘蓝自交不亲和的进一步研究和利用提供了新内容。Self-incompatibility(SI)refers to the characteristic that the pistil of a plant can recognize its own pollen and allogeneic pollen so as to inhibit the germination of its own pollen.PUB(Plant U-Box)proteins play an important role in plant stress resistance and signal transduction.In this study,through the analysis of stigma transcriptome data of Brassica oleracea self-pollination at 0-60 minute(s),a PUB protein coding gene BoPUB9 was screened and up-regulated by self-pollination.The BoPUB9 cDNA sequence was 1368 bp,gDNA of 1720 bp in length,containing one arm repeat domain and one U-box domain.The relative ex-pression levels showed that BoPUB9 gene was expressed in different tissues of Brassica oleracea,and the relative expression level was the highest in sepals,followed by petals,stamens and stigmas,and relatively lower in pollen buds,which was consistent with the results of GUS staining.The relative expression level of BoPUB9 gene was up-regulated rapidly in self-pollination 0-30 minute(s),the expression level after 15 min was more than 20 times that after cross-pollination,and the difference was the biggest after 30 min,the expression level after self-pollination was more than 40 times that after cross-pollination.Subcellular localization revealed that BoPUB9 protein was expressed in both nucleus and cytoplasm,and BoPUB9 protein was successfully induced in E.coli,and the relative molecular weight of 51 kD was consistent with the predicted results.The results of yeast two-hybrid and poll-down indicated that the intracellular domain of SRK interacted with PUB9 protein.It was speculated that BoPUB9 might be a novel gene involved in the process of self-incompatibility in Brassica oleracea,which providing a new content for further re-search and utilization of self-incompatibility in Brassica oleracea.

关 键 词：甘蓝 基因克隆 自交不亲和 表达分析 酵母双杂交 

分 类 号：S635[农业科学—蔬菜学]