马铃薯Y病毒属病毒6K2蛋白叶绿体定位的机制解析  

The mechanism of chloroplast targeting of the 6K2 protein in potyviruses

作　　者：赵金鹏 向小华 张喜峰 杜林林 孙炳剑[1] 汪敏[1] 孙航军 ZHAO Jinpeng;XIANG Xiaohua;ZHANG Xifeng;DU Linlin;SUN Bingjian;WANG Min;SUN Hangjun(College of Plant Protection,Henan Agricultural University,Zhengzhou 450046,China;Haikou Cigar Research Institute,Hainan Provincial Branch of China National Tobacco Corporation,Haikou 571100,China;Baoji Tobacco Company of Shaanxi Province,Baoji 721000,China)

机构地区：[1]河南农业大学植物保护学院,河南郑州450046 [2]中国烟草总公司海南省烟草公司海口雪茄研究所,海南海口571100 [3]陕西省烟草公司宝鸡市公司,陕西宝鸡721000

出　　处：《河南农业大学学报》2024年第6期985-992,共8页Journal of Henan Agricultural University

基　　金：中国烟草总公司海南省公司科技项目(2023002);中国烟草总公司陕西省公司科技项目(KJ-2023-05)。

摘　　要：【目的】明确影响马铃薯Y病毒属病毒6K2蛋白叶绿体定位的结构域。【方法】6K2蛋白分为N端结构域、中间跨膜结构域与C端结构域,分别将6K2蛋白N段结构域、跨膜结构域、C段结构域缺失突变,构建6K2^(PVYΔN)、6K2^(PVYΔC)、6K2^(PVYΔNΔC)、6K2^(TuMVΔN)、6K2^(TuMVΔC)、6K2^(TuMVΔNΔC)突变体。将6K2PVY与6K2^(TuMV)跨膜区的GxxxG基序分别突变,构建6K2PVY GV、6K2^(TuMV)GV突变体,通过激光共聚焦显微镜观察不同6K2-RFP突变体亚细胞定位。将PVY与TuMV编码的6K2蛋白不同结构域重组,构建6K2^(PPT)、6K2^(PTT)、6K2^(TPT)、6K2^(TTP)、6K2^(TPP)、6K2^(PTP)重组蛋白,观察不同6K2-RFP重组蛋白的亚细胞定位。【结果】6K2^(TuMV),突变体6K2^(TuMVΔN)、6K2^(TuMVΔC)、6K2^(TuMVΔN)ΔC、6K2^(PVYΔC)、6K2^(PVYΔNΔC),重组蛋白6K2^(PPT)、6K2^(PTT)、6K2TPT定位于叶绿体。6K2PVY,突变体6K2^(PVYΔN),重组蛋白6K2^(TTP)、6K2^(TPP)、6K2^(PTP)不定位于叶绿体。【结论】马铃薯Y病毒属病毒6K2蛋白的跨膜结构域决定其叶绿体定位,而6K2^(PVY)的C端结构域干扰其叶绿体定位,6K2^(PVY)与6K2^(TuMV)的C端结构域的差异导致了其不同亚细胞定位。【Objective】This study aims to identify the structural domains that determine the chloroplast localization of the 6K2 protein in potyviruses.【Method】The 6K2 protein was segmented into three structural domains:an N-terminal domain,a transmembrane domain,and a C-terminal domain.The N-segment structural domain,the transmembrane structural domain,and the C-segment structural domain of the 6K2 protein were deletion mutated,respectively.We constructed six mutants:6K2^(PVYΔN),6K2^(PVYΔC),6K2^(PVYΔNΔC),6K2^(TuMVΔN),6K2^(TuMVΔC),and 6K2^(TuMVΔNΔC).Mutations targeting the GxxxG motifs within the transmembrane regions of 6K2 PVY and 6K2 TuMV led to the creation of 6K2 PVY GV and 6K2 TuMV GV mutants.Subsequently,we examined the subcellular localization of these distinct 6K2-RFP mutants using laser confocal microscopy.Different domains of 6K2 protein encoded by PVY and TuMV were recombined,and recombinant proteins 6K2^(PPT),6K2^(PTT),6K2^(TPT),6K2^(TTP),6K2^(TPP),6K2^(PTP)were constructed.The subcellular localization of different 6K2-RFP recombinant proteins was then observed.【Result】6K2^(TuMV),along with mutants 6K2^(TuMVΔN),6K2^(TuMVΔC),6K2^(TuMVΔNΔC),6K2^(PVYΔC),6K2^(PVYΔNΔC),and recombinant proteins 6K2^(PPT),6K2^(PTT),and 6K2^(TPT),were localized to chloroplasts.In contrast,the localization of 6K2 PVY,mutant 6K2^(PVYΔN),and recombinant proteins 6K2^(TTP),6K2^(TPP),and 6K2^(PTP)to chloroplasts was not observed.【Conclusion】The transmembrane domain of potyviruses 6K2 protein determines its chloroplast localization.However,the C-terminal structural domain of 6K2 PVY has been observed to interfere with chloroplast localization.Additionally,differences in the C-terminal domains of 6K2 from PVY and TuMV result in distinct subcellular localizations.

关 键 词：马铃薯Y病毒属 6K2蛋白 叶绿体 跨膜结构域 亚细胞定位 

分 类 号：S432.1[农业科学—植物病理学]