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利用已知的RSG6基因序列和GenBank中提供的RSG6前导序列设计引物 ,通过基因组DNA的PCR扩增得到长度为 1 4 0 8bp和 1 1 73bp的两个RSG6启动子片段PB、PS,测定序列分析发现 ,PB、PS分别位于水稻第 1 0和第 4染色体上 ,PB、PS序列之间具有较高的同源性 ,且都含有大量的启动子元件。通过设计带有酶切位点的引物扩增出PB和PS的各两条缺失片段PB1、PB2、PS1、PS2 ,与质粒 pBI2 2 1连接后得到中间载体pBI2 2 1 PB1、pBI2 2 1 PB2、pBI2 2 1 PS1、pBI2 2 1 PS2 ,再与质粒pBIN1 9连接构建出双元表达载体pBIN GUSB1、pBIN GUSB2、pBIN GUSS1、pBIN GUSS2 ,转化农杆菌EHA1 0 5 ,感染烟草叶片和烟草花粉 ,瞬时表达显示缺失片段PB1、PB2、PS1、PS2均具有启动子功能 ,且PB1、PS1的启动效率较PB2、PS2高
Primers were designed based on the known RSG6 gene sequence and the RSG6 leader sequence provided in GenBank. Two RSG6 promoter fragments PB and PS were obtained by PCR amplification of genomic DNA with length of 140 bp and 1173 bp. Sequencing analysis It was found that PB and PS were located on rice chromosomes 0 and 4, respectively, and had high homology between PB and PS sequences and contained a large number of promoter elements. The two deletion fragments PB1, PB2, PS1 and PS2 of PB and PS were amplified by designing a primer with restriction enzyme site, and then ligated with plasmid pBI2 2 1 to obtain the intermediate vectors pBI2 2 1 PB1, pBI2 2 1 PB2, pBI2 2 1 PS1 and pBI2 2 1 PS2 were constructed and ligated with plasmid pBIN1 9 to construct binary expression vectors pBIN GUSB1, pBIN GUSB2, pBIN GUSS1 and pBIN GUSS2, transformed into Agrobacterium tumefaciens EHA1 05 and infected with tobacco leaves and tobacco pollen for transient expression The deletion fragments PB1, PB2, PS1 and PS2 all showed promoter function, and the priming efficiency of PB1 and PS1 was higher than that of PB2 and PS2