摘要
将RDV微核心蛋白基因S9克隆到酵母表达载体pGBKT7中,转入AH109酵母细胞,转化子可以在SD/Trp-His-Ade-多营养缺陷固体培养基上正常生长,证明RDV P9在酵母中具有转录激活活性,运用β-半乳糖苷酶的活性分析对重组质粒转化子的转录激活程度做了定量分析,发现与正对照相比,转化pGBK-S9的酵母菌中β-半乳糖苷酶活性可达到正对照的40%以上。构建了含有gusA报告基因的植物表达载体用来分析P9在植物中的转录激活活性,实验结果表明,融合GAL4 DB的P9蛋白可以在植物体内激活报告基因的表达,Western印迹法分析表明了P9蛋白在酵母和植物中均可以表达。证明了在植物体内RDV P9蛋白同样能够激活基因的转录,暗示该蛋白可能在病毒的侵染和复制过程中参与调控病毒或寄主基因的转录表达。
The RDV gene segment S9 was cloned into yeast expression vector pGBKT7 for growth test assay, pGBK-S9 plasmid was transformed into host strain AH109, and could grow on synthetic medium lacking tryptophan, histidine, and adenine （SD/-Trp/-His/-Ade）. Results showed that RDV P9 performed the transcriptional activation in yeast. To further examine transcriptional activation of P9 in yeast, o-nitrophenyl β-D-galactopyanoside （ONPG） was used as a substrate for quantitative assay. Based on the growth ability test, the quantitative analysis strongly supported that RDV P9 showed transcriptional activation in yeast. To verify the transcriptional activation of P9 in plants, the transiently expressed system was conducted by using β-glucuronidase （GUS） as a reporter. The results indicated that strong GUS activities were visible in tissues. Western blotting analysis proved the expression of P9 in yeast and N. benthamiana plants. These results suggested that RDV P9 might play an important role in regulation of virus and genome transcription for the benefit of virus infection.
出处
《中国农业科技导报》
CAS
CSCD
2007年第3期61-65,共5页
Journal of Agricultural Science and Technology
基金
国家自然基金重点项目水稻矮缩病毒(RDV)编码RNA沉默抑制因子作用机制的研究(30530030)
国家973项目农作物抗病反应的分子基础及基因网络(2006CB101903)资助
