烟草rbcS启动子的克隆与活性鉴定  

Cloning of tobacco rbcS promoter and determination of its activity

作　　者：孙家利[1,2] 闫晓红[2] 王力军[2] 时向东 魏文辉[2] 

机构地区：[1]河南农业大学国家烟草栽培生理生化研究基地,郑州文化路95号450002 [2]中国农业科学院油料作物研究所基因组学与分子生物学研究室,武汉430062

出　　处：《中国烟草学报》2010年第3期80-85,共6页Acta Tabacaria Sinica

基　　金：国家自然科学基金项目(30671312);武汉市青年科技晨光计划项目(200750731300)

摘　　要：为了有效启动外源基因在烟草叶部特异性表达并改良烟叶品质,采用PCR技术从烟草栽培种NC89基因组中分离了rbcS启动子序列,扩增片段长度979bp。PlantCARE序列分析表明,该序列中含有启动子特征的保守序列及十几种光应答元件,与已报道序列的相应区域同源性达99%。将其与GUS融合蛋白基因串连,构建了植物表达载体prbcS-121,通过根癌农杆菌介导的烟草叶盘转化法,将pBI121及prbcS-121转化烟草。对转基因烟草植株中GUS活性测定结果表明,烟草rbcS启动子具有叶部组织特异性,且在叶部启动基因表达能力较CaMV35S启动能力强,从而为外源基因在转基因烟草叶片中的高效表达提供了新的工具,为后续目的基因能在烟草叶部发生组织特异性和光诱导性表达奠定了基础。In order to promote specific expression of alien gene in leaf tissue of tobacco and improve leaf quality,sequence of ribulose-1,5-bisphosphate carboxylase small subunit （rbcS） promoter was isolated from tobacco cultivar NC89 genome by PCR with amplified segment size of 979bp. PlantCARE sequence analysis showed that the sequence contained conservative sequence of promoter,and also a dozen light-responsive elements. There was 99% sequence identity with reported corresponding sequence. A recombinant expression vector prbcS-121 was constructed with rbcS promoter and GUS gene,together with pBI121,transformed into tobacco by Agrobacterium tumefaciens-mediated transformation method. The determinations of the GUS activities in ttransgenic tabacco plants indicated that rbcS promoter can drive the expression of GUS gene in leaves of transgenic plants,and the expression level of rbcS-GUS fusion gene was significantly stronger than that of CaMV35S-GUS fusion gene. A new tool was offered for high-level expression of foreign gene in transgenic plant which can achieve tissue-special and light-regulated expression of interesting gene.

关 键 词：烟草 rbcS启动子 rbcS-GUS融合基因 GUS活性 转基因烟草 

分 类 号：S572[农业科学—烟草工业]