一个水稻鞘叶特异表达启动子的克隆及功能鉴定  

Cloning and Functional Analysis of a Promoter with Tissue Specific Expression in Rice (Oryza sativa L ssp. japonica) Sheath and Leaf

作　　者：颜彦[1] 林拥军[1] 

机构地区：[1]华中农业大学作物遗传改良国家重点实验室,武汉430070

出　　处：《农业生物技术学报》2013年第7期757-763,共7页Journal of Agricultural Biotechnology

基　　金：国家科技重大专项"抗虫转基因水稻新品种培育"(No.2011ZX08001-001)

摘　　要：目前在基因工程中应用最广的为组成型启动子,但是组成型启动子驱动外源基因在各组织中持续恒定的表达可能引起植物发育迟缓等问题,因此克隆来源于作物本身的组织特异型启动子逐渐受到重视。根据本实验室基因芯片数据库找到一个在水稻绿色组织特异表达的基因(TIGR Locus:LOC_Os06g21110),用 PCR 技术从水稻明恢 63(Oryza sativa L ssp. indica)基因组中克隆得到其上游启动子命名为GSP (green-tissue specific promoter),长度为1 951 bp。将GSP与带有β-glucuronidas(gus)报告基因的的植物表达载体连接,经农杆菌(Agrobacterium)介导转化水稻中花11 (O. sativa L ssp. japonica)成熟种子胚诱导的愈伤组织,获得转基因水稻阳性植株,通过组织化学染色法证明,GSP是一个叶鞘和叶片特异表达启动子。对其进行5'端缺失分析,构建了7个缺失载体,通过验证缺失载体的表达谱,证明592 bp长度的启动子就足以维持鞘叶特异表达模式,初步鉴定出了该启动子的核心功能区域。本研究所克隆出的GSP 启动子是一个未曾报道过的鞘叶特异表达启动子,基因工程中利用此类启动子可以在改良水稻性状的同时减少对水稻生理方面的副作用,有良好的应用价值。Most of the well-characterized promoters that are currently available confer constitutive expression in genetic engineering, however, this expression model may cause plant growth retardation and other problems, so cloning novel tissue-speci fi c promoter is needed. Based on microarray data, we identified a rice green-tissue specific expression gene （TIGR Locus： LOC_Os06g21110）, a promoter named green-tissue specific promoter（GSP） with 1 951 bp in length of the gene was cloned by PCR from the genomic DNA office Minghui 63（0. sativa L ssp. #Mica）. Promoter GSP was further fused with t-glucuronidas （gus） reporter gene and transformed into rice Zhonghua 1 l（Oryza sativa L ssp. japonica） callus of mature seed through Agrobacterium-mediated transformation. The expression pattern of GSP was identified in transgenic rice plants by histochemical staining. The result verified that GSP was a sheath and leaf specific expression promoter. Then 5＇ end different length deletions of GSP were fused with gus gene and were transformed into rice respectively. The result of deletion analyses indicated that 592 bp length promoter fragment was sufficient for maintain the sheath and leaf specific expression model, and also identified core functional areas of GSP preliminarily. GSP is a sheath and leaf specific promoter which has not been reported, utilizing tissue specific promoters like GSP will subject to rice improvement and limit the potential of unintended impacts on plant physiology, so it has a good application value in transgenic rice.

关 键 词：水稻 启动子 组织特异表达 GUS报告基因 缺失分析 

分 类 号：Q943.2[生物学—植物学]