摘要
在通过酵母双杂交文库获得了与钙响应蛋白BoSPx相互作用蛋白BoPID的基础上,对BoPID的编码基因进行了克隆、时空特异性表达分析及筛选与其互作的蛋白,以期为BoSPx通过BoPID参与自交不亲和性分子过程提供依据。结果表明,BoPID包含2个外显子和1个内含子,编码439个氨基酸。激酶磷酸化预测分析显示具有激酶活性;系统进化和共线性分析显示BoPID具有较强的植物种属特异性,与芜菁、拟南芥和甘蓝型油菜等十字花科植物聚类在一起,BoPID与AtPID在染色体水平上高度同源。组织特异性表达分析显示BoPID在柱头中的表达量高于花器官中的其他部位。荧光定量PCR分析表明BoPID在甘蓝柱头自花授粉15 min显著上调表达,而后下调表达。启动子元件分析发现BoPID含有ABA、IAA、脱落酸、茉莉酸、水杨酸和胁迫响应等多个应答元件。BoPID蛋白定位于细胞膜和细胞质中,可能是一种泊位于膜上并突触于胞质中的双栖蛋白。酵母双杂交和GST-Pulldown结果表明BoPID与BoCML12、BoCaM2、BoPIN1能够发生相互作用。BoPID可能是BoSPx与Bo CML12、BoCaM2之间相互作用的桥梁蛋白,共同通过生长素调节钙响应的方式参与了自交不亲和分子过程。
The BoPID was found as one of the interactive protein of BoSPx through yeast two-hybrid screening assay in Brassica oleracea.Its characters such as coding sequence,spatiotemporal expression and interactive proteins were further analyzed,with the purpose of studying its possible role in interacting with the calcium-responsive protein BoSPx in the process of self-incompatibility.The results showed that BoPID encodes 439 amino acids and contains two exons and one intron.BoPID may have kinase activity based on the kinase phosphorylation prediction analysis of its sequence,its sequence is highly homologous with At PID at the chromosome level and has strong species specificity when clustering with cruciferous plants such as turnip,Arabidopsis and Brassica napus in the phylogeny and collinearity analysis.Additionally,its promoter area contains multiple response elements such as ABA,IAA,abscisic acid,jasmonic acid,salicylic acid and stress response.The spatiotemporal expression analysis indicated that BoPID expressed higher in the stigma than that in other parts of floral organs,while it was up-regulated after 15 minutes of self-pollination and then down-regulated.In the analysis of subcellular localization,we found BoPID protein localized in the cell membrane and cytoplasm,indicating that it might be an amphibious protein and anchored on the membrane and synapses in the cytoplasm.We further found the proteins of BoCML12,BoCaM2 and BoPIN1 interact with BoPID in the assays of yeast two-hybrid and GST-Pull down.Based on these results,we reason that BoPID might play a role in regulating calcium response to auxin through interacting with BoSPx,BoCML12 and BoCaM2 in the process of self-incompatibility.
作者
张贺翠
王玉奎
左同鸿
刘倩莹
张以忠
胡燈科
谢琴琴
朱利泉
ZHANG Hecui;WANG Yukui;ZUO Tonghong;LIU Qianying;ZHANG Yizhong;HU Dengke;XIE Qinqin;ZHU Liquan(College of Agronomy and Biotechnology,Southwest University,Chongqing 400715,China)
出处
《园艺学报》
CAS
CSCD
北大核心
2021年第6期1123-1134,共12页
Acta Horticulturae Sinica
基金
国家自然科学基金项目(31572127)。
