水稻OsGSTLc启动子的分离和鉴定

Isolation and identify of the rice OsGSTLc promoter

关键词:  OsGSTLc  诱导  缺失分析  启动子  水稻

Key words:  OsGSTLc  induction  deletion analysis  promoter  rice

摘要:

半定量RT-PCR分析表明,OsGSTLc在水稻根中的表达受绿磺隆的诱导。从水稻基因组中分离到的OsGSTLc读码框上游2 2171 bp序列,在起始密码ATG上游-86 bp处有CAAT-box,但在CAAT-box与读码框之间没有典型的TATA-box。因此,OsGSTLc启动子是无TATA框启动子。将OsGSTLc启动子5’-端系列缺失后,分别与GUS报告基因融合,获得GSTL2171::GUS、GSTL1761::GUS、GSTL962::GUS和GSTL525::GUS表达载体,利用农杆菌介导转化水稻,获得转基因水稻,均能启动下游GUS报告基因的表达。氯磺隆处理后,转入GSTL2171::GUS、GSTL1761::GUS和GSTL962::GUS的水稻植株根部的GUS活性明显增加。氯磺隆诱导的应答元件在-962～-525 bp的范围内。

Abstract:

The expression of OsGSTLc in rice roots was induced by chlorsulfuron through semi-quantitative RT-PCR analysis demonstrated. A 2 171 bp upstream sequence of the translation start codon ATG of OsGSTLc gene was isolated from the genomic DNA of rice,which contained a putative CAAT-box at position -86 bp upstream of ATG,but there wasn’t TATA-box between the CAAT-box and ATG. Therefore,OsGSTLc promoter was a TATA-less promoter. To define the core promoter sequence,a series of 5’ truncation derivatives of GST2171 were fused to a GUS reporter gene to construct GSTL2171::GUS,GSTL1761::GUS,GSTL962::GUS and GSTL525::GUS,respectively. The four fusion genes were introduced into rice plants by Agrobacterium-mediated transformation,all promoter fragments with 5’-deletion drived successfully the expression of GUS report gene. Quantitative fluorescence assays showed that the GUS activities in the roots of GSTL2171::GUS,GSTL1761::GUS and GSTL962::GUS transgenic rice seedlings were upregulated by chlorsulfuron. The transcriptional activation element of chlorsulfuron may be located between positions -962 and -525.The expression of OsGSTLc in rice roots was induced by chlorsulfuron through semi-quantitative RT-PCR analysis demonstrated. A 2 171 bp upstream sequence of the translation start codon ATG of OsGSTLc gene was isolated from the genomic DNA of rice,which contained a putative CAAT-box at position -86 bp upstream of ATG,but there wasn’t TATA-box between the CAAT-box and ATG. Therefore,OsGSTLc promoter was a TATA-less promoter. To define the core promoter sequence,a series of 5’ truncation derivatives of GST2171 were fused to a GUS reporter gene to construct GSTL2171::GUS,GSTL1761::GUS,GSTL962::GUS and GSTL525::GUS,respectively. The four fusion genes were introduced into rice plants by Agrobacterium-mediated transformation,all promoter fragments with 5’-deletion drived successfully the expression of GUS report gene. Quantitative fluorescence assays showed that the GUS activities in the roots of GSTL2171::GUS,GSTL1761::GUS and GSTL962::GUS transgenic rice seedlings were upregulated by chlorsulfuron. The transcriptional activation element of chlorsulfuron may be located between positions -962 and -525.