水稻OsPIN1a基因体外表达及蛋白磷酸化活性位点分析  

Expression in vitro of OsPIN1a Gene and Analysis of Phosphorylation Sites

作　　者：王尉[1,2] 郭宁[1] 徐明照[1] 田海燕[2] 石胜友[2] 

机构地区：[1]安徽农业大学生命科学学院,安徽合肥230036 [2]中国热带农业科学院南亚热带作物研究所,广东湛江524091

出　　处：《热带作物学报》2014年第8期1528-1532,共5页Chinese Journal of Tropical Crops

基　　金：安徽省自然科学基金(No.1308085QC50);高校省级优秀青年人才基金项目(No.2012SQRL057)

摘　　要：输出载体OsPIN1a是水稻PIN-FORMED(PIN)的家族成员之一,参与调节生长素在植物组织中的不均衡分布。然而,OsPIN1a蛋白的功能结构域如何调节激素的极性运输仍需进一步探讨。本研究拟以日本晴水稻为材料,利用OsPIN1a基因的非编码区设计引物,特异地克隆目的基因,并借助Quickchange XL site-directed mutagenesis技术分别构建单突变[pET30-OsPIN1a(Ser233/Ala)和pET30-OsPIN1a(Ser254/Ala)]及双突变[pET30-OsPIN1a(Ser233/Ala和Ser254/Ala)]表达载体,通过体外蛋白表达和磷酸化活性分析,明确了OsPIN1a蛋白水解结构域中Ser-233和Ser-254是丝氨酸/苏氨酸蛋白激酶(PINOID,PID)的磷酸化活性位点,此结果为进一步研究生长素的极性运输机制提供参考。As one of PIN-FORMED（PIN） efflux carriers, OsPINla is involved in regulating auxin distributions in plant tissues. However, how these functional sites of OsPINla regulate hormone distribution still need to be further analyzed. In our study, OsPINla gene was isolated from Oryza sativa L. cv. Nipponbare using the specific primers, designed according to untranslated regions. To explore the phosphorylation sites of OsPINla in proteolytic structures, the expression vectors of single mutation[pET30-OsPINla（Ser233/Ala） and pET30-OsPINla（Ser254/Ala）] and double mutation[pET30-OsPINla（Ser233/Ala and Ser254/Ala）] were constructed by quickchange XL site-directed mutagenesis technique, respectively. OsPINla was expressed and analyzed by the expression system in vitro. Two phosphorylation sites of Ser-233 and Ser-254 can be phosphorylated by a serine-threonine protein kinase（PINOID, PID）. The results provide a reference for further studying the mechanism of auxin polar transport.

关 键 词：水稻 OsPIN1a 蛋白表达 磷酸化 

分 类 号：S511[农业科学—作物学] Q78[生物学—分子生物学]