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2025年4月4日 星期五
  2015, Vol. 23 Issue (11): 1410-1420    
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
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
张超1,高芳銮1,郑璐平2,丁作美3,杨振1,李阳1,吴建国1,吴祖建1
1. 福建农林大学植物病毒研究所
2. 福建农林大学教育部重点实验室
3.
Gain of Rice (Oryza sativa) DEAD-box RNA Helicase (OsRH37) Overexpression and RNAi Transgenic Plants and Analysis of Its Subcellular Localization
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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OsRH37的CDS全长序列。序列比对发现,OsRH37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OsRH37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OsRH37超表达和RNAi T0转基因水稻阳性植株。sqRT-PCR分析表明,与野生型日本晴水稻中OsRH37表达量相比,OsRH37超表达T1转基因水稻中OsRH37表达量均明显上调,而OsRH37 RNAi转基因水稻中OsRH37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-OsRH37,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,OsRH37蛋白定位于细胞核、染色质核仁和细胞质中的点状结构。OsRH37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。
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张超
高芳銮
郑璐平
丁作美
杨振
李阳
吴建国
吴祖建
关键词 DEAD-box RNA解旋酶(RH)OsRH37超表达RNAi亚细胞定位    
Abstract: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 OsRH37, encoding a putative DEAD-box RNA helicase, was cloned. The amino acid sequence of OsRH37 was analyzed by bioinformatics method and it was indicated that the amino acid sequence identity between OsRH37 and 3 other Arabidopsis thaliana DEAD-box RNA helicases including AtRH17, 37 and 52 was>70%, whereas the amino acid sequence identity between OsRH37 and DEAD-box RNA helicases from different animals was generally<60%. The overexpression vector and RNAi vector of OsRH37 were constructed, and by using rice genetic transformation system the overexpression and RNAi transgenic rice plants of OsRH37 were obtained. Six lines (66.7%) among 9 lines of OsRH37 overexpressed transgenic rice and 13 lines (92.9%) among 14 lines of OsRH37 RNAi transgenic rice were identified to be positive, respectively. The relative expression level of OsRH37 among wild type, overexpression lines, and RNAi lines were detected by sqRT-PCR. The results indicated that compared with the wild type (WT), the expression level of OsRH37 in overexpression lines was obviously up-regulated while that in RNAi lines was significantly down-regulated. To observe the subcellular localization pattern of OsRH37, the stop codon TAG deleted CDS was constructed into the transient expression vector pEarleyGate102, harboring a cyan fluorescence protein (CFP), by Gateway technology and the leaves of Nicotiana benthamiana agroinfiltrated with CFP-OsRH37 were observed under confocal microscope at 48 h post infiltrated. The results of subcellular localization analysis suggested that OsRH37 fused with CFP was mostly localized at nucleus, karyosome and cytoplasmic granular structures. The bioinformatics analysis, obtained transgenic rice plants and subcellular localization analysis of OsRH37 can provide foundational insights for the important roles of OsRH37 in the processes of the rice growth, development and stress responses.
Key wordsDEAD-box RNA helicase (RH)    OsRH37    Overexpression    RNAi    Subcellular localization
收稿日期: 2015-06-26      出版日期: 2015-10-05
基金资助:国家自然科学基金;福建省自然科学基金
通讯作者: 吴建国     E-mail: wujianguo81@126.com
引用本文:   
张超 高芳銮 郑璐平 丁作美 杨振 李阳 吴建国 吴祖建. 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[J]. , 2015, 23(11): 1410-1420.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I11/1410
 
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