水稻GOS2基因高活性启动子的分离及其在叶片中的特性鉴定  

Isolation and Characterization of an Efficient GOS2 Promoter in Rice(Oryza sativa L.) Leaf Tissue

作　　者：张利东[1] 朱骞[1] 董陈文华 张树林[1] 伍腾飞[1] 熊海波[1] 张小玲[1] 吕永刚[2] 吴超[1] 李伟[1] 陈丽娟[1,3] 李东宣[1,3] 

机构地区：[1]云南农业大学稻作研究所,昆明650201 [2]云南省农业科学院,昆明650205 [3]云南省高校滇型杂交粳稻分子育种重点实验室,昆明650201

出　　处：《分子植物育种》2015年第1期32-38,共7页Molecular Plant Breeding

基　　金：NSFC-云南联合基金重点项目(U1136604);国家重点基础研究973项目(2011CB1-00401);云南省基金重点项目(2006C006Z)共同资助

摘　　要：高活性启动子在基因时空表达调控方面有着重要的作用,利用高效率启动子调控抗性目的基因特异性表达,不仅可以提高作物抗胁迫能力,而且对改良品种方面具有重要的意义。迄今为止,有关水稻翻译起始因子GOS2基因(eukaryotic translation initiation factor 1b)启动子p GOS2启动效率鉴定的报道还很有限,尤其是与Ca MV35S双拷贝启动子的启动效率评估的研究还未见报道。本研究旨在利用PCR和生物信息学等技术,分离及解析水稻GOS2启动子特性,构建p GOS2::GUS双元表达载体,使用农杆菌介导法转化水稻胚性愈伤组织并获得转基因水稻植株,并通过PCR检测与GUS组织化学分析,评估p GOS2在转基因水稻叶片组织中的效率。生物信息学分析结果表明:所克隆的GOS2启动子,序列全长为3 155 bp,具有真核生物典型的一些顺式元件,如TATA-box、AGA-motif、CAAT-box以及GCGC-repeat等;GOS2启动子核心序列位于-43 bp^+4 bp区,得分是0.97。GUS组织化学分析结果显示:阴性对照组未发现GUS信号,而转基因实验组则表现为不同程度的GUS活性,p GOS2启动效率远远高于单双拷贝的Ca MV35S启动子。本研究结果证实了GOS2启动子在水稻叶片组织中的活性远远地高于加强型Ca MV35S启动子,为今后水稻分子育种及相关启动子的开发与筛选提供了一种可行的方法和新的视野。Highly active promoter plays a crucial role in gene expression and regulation in time and space. Specific-expression of targeted resistant genes using high efficiency promoter, not only can improve crop resistance to biotic-abiotic stresses, but also has important significance in crop improvement. To date, the reports are very limited on identification efficiency of rice GOS2 gene （translation initiation factor lb） promoter pGOS2, especially, the comparison of efficiency between double copies of CaMV35S and single copy of pGOS2 have not been reported. This study was aimed at： isolation and characterization of rice GOS2 promoter by using PCR method and bioinformatics technology; construction of the pGOS2：：GUS binary expression vector; transformation of riceembryogenic callus with agrobacterium-mediated method, identification of positive transgenic rice plants by PCR and GUS histochemical assays, and evaluation of the efficiency ofpGOS2 promoter in leaf tissue of transgenic rice plant. Bioinformatics analysis showed that the GOS2 promoter with full-length of 3 155 bp, contained some typical cis-elements of eukaryotes, such as TATA-box, AGA-motif, CAAT-box and GCGC-repeat, etc., exhibiting a promoter core sequence at -43 bp-＋4 bp with a score of 0.97. The GUS histochemical assays indicated that in the negative control groups did not appear GUS signal, on the contrary, transgenic lines all came out different levels of GUS activity, pGOS2 was far more efficient than single and double CaMV35S promoter. The results of this research confirmed the activity of GOS2 promoter was much higher than the CaMV35S promoter in rice leaf tissue, which would add a feasible approach and a new insight for rice molecular breeding, exploiting and screening of related promoter in the future.

关 键 词：水稻(Oryza SATIVA L.) GOS2 启动子 克隆 GUS组织化学分析 

分 类 号：S511[农业科学—作物学]