植物病毒载体介导的sMMO-C在蚕豆植物中的表达

Expression of the sMMO-C in the Broad Bean Plant by the Plant Virus Vector

构建了可溶性甲烷单加氧酶C亚单位(soluble methane monoxygenase-C component,sMMO-C)基因和绿色荧光蛋白-S65T(green fluorescence protein,GFP-S65T)基因的融合基因sMMO-C/GFP-S65T。将融合基因sMMO-C/GFP-S65T克隆到三叶草黄脉病毒(glover yellow vein virus,ClYVV)侵染性植物表达载体pClYVV/CP/W的NIb/CP基因之间,构建了侵染性重组病毒克隆pCV-mmoC/GFP-S65T。RT-PCR检测结果表明,sMMO-C/GFP-S65T在子代病毒基因组中获得了转录。用GFP抗体进行蛋白质杂交(western blotting,WB),检测到25kDa左右的GFP蛋白,这一结果表明GFP蛋白在被pCV-mmoC/GFP-S65T侵染的寄主植物中获得了表达,并被病毒自身编码的NIa蛋白酶从病毒聚合蛋白及sMMO-C/GFP-S65T融合蛋白中自动切断。可见,sMMO-C亚单位蛋白在植物中获得了表达,因在sMMO-C/GFP-S65T中sMMO-C位于GFP上游。

sMMO-C/GFP-S65T,the fusion gene of the sMMO-C（Soluble methane monoxygenase-C component）and the GFP-S65T（Green fluorescence protein-S65T）gene was constructed,and the fusion gene sMMO-C/GFP-S65T was cloned between nuclear inclusion b（NIb） and coat protein（CP） of infectious viral vector pClYVV/CP/W derived from clover yellow vein virus（ClYVV） of potyvirus.The recombination infectious plasmids were constructed and named pCV-mmoC/GFP-S65T.The genetic stability of the pCV-mmoC/GFP-S65T carrying sMMO-C/GFP-S65T fusion gene was proved by RT-PCR examination.The about 25kDa GFP protein was detected by western blot analysis using antibodies against the GFP protein.The results showed that these GFP gene was expressed in host infected by recombined pCV-mmoC/GFP-S65T,and cleaved automatically from virus poly-proteins and sMMO-C/GFP-S65T fusion proteins by self-coding NIa protease.Meanwhile,according to the results above,it is possible to extrapolate that the sMMO-C component protein has also been expressed in the plant,because sMMO-C gene is located upstream of GFP-S65T gene in sMMO-C/GFP-S65T fusion gene.