柑橘黄化脉明病毒的TGB基因生物信息学分析及亚细胞定位

Bioinformatics and Subcellular Localization on Citrus yellow vein clearing virus-Triple Gene Block

柑橘黄化脉明病毒(Citrus yellow vein clearing virus,CYVCV)是一种正义单链RNA病毒。其基因组含有6个开放阅读框(ORF),其中ORF2、ORF3和ORF4组成了三基因连锁结构(triple gene block,TGB)。以感染CYVCV的尤力克柠檬[Citrus limon(L.)Burm.f.]植株的总RNA为模板,扩增和克隆了CYVCV的TGB基因,并开展了其编码蛋白的理化性质和分子特性等生物信息学分析;进而通过In-Fusion~?技术构建了TGB各基因的亚细胞定位载体,经农杆菌介导转化洋葱表皮细胞并在荧光显微镜下进行亚细胞定位观察。生物信息学分析结果表明,CYVCV-TGB具有与Potexvirus属病毒TGB相似的理化性质和分子特性,可能参与病毒在寄主中的运动且需要外壳蛋白的参与。亚细胞定位结果显示,TGBp1定位于细胞壁(膜)上,TGBp2和TGBp3主要定位于细胞壁(膜),少量呈星点状定位于细胞内。研究结果为揭示CYVCV在寄主中的运动机理奠定了基础。

Citrus yellow vein cleansing virus （CYVCV） is a simple positive-sense RNA virus. Its genome contains six open reading frames （ORFs）, among which ORF2, ORF3 and ORF4 constitute a triple gene block （TGB） . The TGB gene of CYVCV was amplified and cloned by using the total RNA of infected Eureka lemon [ Citrus limon （L.） Burro. F.1. B ioinformatic analysis of TGB encoded proteins were carried out with the physicochemical property and molecular characteristics. The TGB gene subcellular localization vector were constructed through the In-Fusion technology and transformated to onion epidermal cells mediated by Agrobacterium tumefaciens. Subcellular localization was then observed under fluorescence microscope. Bioinformatics analysis results showed that the physicochemical properties and molecular characteristics of CYVCV-TGB were similar to that of the Potexvirus, and the TGB may involved in virus movement in the host. Subcellular localization results showed that TGBp 1 was located on the cell wall （cell membrane）, while TGBp2 and TGBp3 were mainly located on the cell wall （cell membrane）, with a small amount located in the cell. These results laid the foundation of revealing the CYVCV movement mechanism.