水稻DEAD-box RNA解旋酶OsRH37超表达和RNAi转基因水稻的获得及其亚细胞定位分析  

Gain of Rice(Oryza sativa) DEAD- box RNA Helicase(OsRH37)Overexpression and RNAi Transgenic Plants and Analysis of Its Subcellular Localization

作　　者：张超[1] 高芳銮[1] 郑璐平[1] 丁作美 杨振[1] 李阳[1] 吴建国[1] 吴祖建[1] 

机构地区：[1]福建农林大学植物病毒研究所/福建省植物病毒学重点实验室,福州350002

出　　处：《农业生物技术学报》2015年第11期1410-1420,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31272018和No.31201491);福建省自然科学基金(No.2013J01089);福建省高校杰出青年科研人才计划(No.JA3091)

摘　　要：DEAD-box RNA解旋酶(RNA helicase,RH)普遍存在于绝大多数的生物体中,参与到RNA新陈代谢的各个方面。本研究从日本晴水稻(Oryza sativa L.japonica.cv.Nipponbare)叶片RNA中成功克隆DEAD-box RNA解旋酶基因Os RH37的CDS全长序列。序列比对发现,Os RH37的氨基酸序列与拟南芥(Arabidopsis thaliana)DEAD-box RNA解旋酶17、37和52的氨基酸序列一致性>70%,与来自于不同动物体内DEAD-box RNA解旋酶的氨基酸序列一致性普遍<60%。构建了Os RH37的水稻超表达和RNAi干扰载体,通过水稻遗传转化体系成功转化水稻获得Os RH37超表达和RNAi T0转基因水稻阳性植株。sq RT-PCR分析表明,与野生型日本晴水稻中Os RH37表达量相比,Os RH37超表达T1转基因水稻中Os RH37表达量均明显上调,而Os RH37 RNAi转基因水稻中Os RH37的表达量均显著下调。构建青色荧光蛋白的瞬时表达载体CFP-Os RH37,经农杆菌(Agrobacterium tumefaciens)接种导入本氏烟(Nicotiana benthamiana)叶片表皮细胞,激光共聚焦显微观察结果显示,Os RH37蛋白定位于细胞核、染色质核仁和细胞质中的点状结构。Os RH37的生物信息学分析、超表达和RNAi转基因水稻的获得以及亚细胞定位分析将为研究其在水稻生长发育和胁迫反应中发挥的重要作用提供线索。DEAD-box RNA helicase（RH） ubiquitously exists in most organisms and participates, if not all,steps of cellular RNA metabolism. Generally, containing 9 highly conserved motifs is an important standard to identify a DEAD- box RNA helicase. In the present study, the CDS of a rice（Oryza sativa L. japonica. cv.Nipponbare） gene Os RH37, encoding a putative DEAD- box RNA helicase, was cloned. The amino acid sequence of Os RH37 was analyzed by bioinformatics method and it was indicated that the amino acid sequence identity between Os RH37 and 3 other Arabidopsis thaliana DEAD- box RNA helicases including At RH17, 37 and 52 was〉70%, whereas the amino acid sequence identity between Os RH37 and DEAD- boxRNA helicases from different animals was generally60%. The overexpression vector and RNAi vector of Os RH37 were constructed, and by using rice genetic transformation system the overexpression and RNAi transgenic rice plants of Os RH37 were obtained. Six lines（66.7%） among 9 lines of Os RH37 overexpressed transgenic rice and 13 lines（92.9%） among 14 lines of Os RH37 RNAi transgenic rice were identified to be positive, respectively. The relative expression level of Os RH37 among wild type, overexpression lines, and RNAi lines were detected by sq RT- PCR. The results indicated that compared with the wild type（WT）, the expression level of Os RH37 in overexpression lines was obviously up-regulated while that in RNAi lines was significantly down-regulated. To observe the subcellular localization pattern of Os RH37, the stop codon TAG deleted CDS was constructed into the transient expression vector p Earley Gate102, harboring a cyan fluorescence protein（CFP）, by Gateway technology and the leaves of Nicotiana benthamiana agroinfiltrated with CFP-Os RH37 were observed under confocal microscope at 48 h post infiltrated. The results of subcellular localization analysis suggested that Os RH37 fused with CFP was mostly localized at nucleus, karyosome and cytoplasmic granular structures. The bioinformatics analy

关 键 词：DEAD-BOX RNA解旋酶(RH) OsRH37 超表达 RNAI 亚细胞定位 

分 类 号：S511[农业科学—作物学]