College of Plant Protection, Henan Agricultural University, Zhengzhou, 450002, China.
Zhengzhou Fruit Research Institute, Chinese Academy of Agriculture Sciences, Zhengzhou, 450009, China.
Virol J. 2018 May 24;15(1):93. doi: 10.1186/s12985-018-1004-9.
Cucurbit chlorotic yellows virus (CCYV), a bipartite crinivirus, causes chlorotic leaf spots and yellowing symptoms on cucurbit leaves. We previously developed an infectious clone of CCYV. Limited work has been conducted on the construction of a crinivirus green fluorescence protein (GFP) expression vector to date.
We constructed a CCYV GFP expression vector using the "add a gene" strategy based on CCYV RNA2 cDNA constrcut. Three resultant clones, pCCYVGFP, pCCYVGFP, and pCCYVGFP were constructed with different promoters used to initiate GFP and CP expression. At 25 dpi GFP fluorescence was detectable not only in leaf veins but also in the surrounding cells. pCCYVGFP-infected cucumber leaves exhibited cell spread at 25 dpi, whereas pCCYVGFP and pCCYVGFP were mainly found in single cells. Further observation of pCCYVGFP GFP expression at 30 dpi, 40 dpi, and 50 dpi showed phloem-limited localization in the systemic leaves.
We developed of a CCYV GFP expression vector that will be useful for further study of CCYV movement in cucurbits.
葫芦科褪绿黄斑病毒（CCYV）是一种二分体呼肠孤病毒，会导致葫芦科叶片出现褪绿黄斑和黄化症状。我们之前开发了 CCYV 的传染性克隆。迄今为止，关于构建呼肠孤病毒绿色荧光蛋白（GFP）表达载体的工作有限。
我们使用基于 CCYV RNA2 cDNA 构建的“添加基因”策略构建了 CCYV GFP 表达载体。使用不同的启动子来启动 GFP 和 CP 表达，构建了三个克隆，pCCYVGFP、pCCYVGFP 和 pCCYVGFP。在 25dpi 时，不仅在叶脉中，而且在周围细胞中都可以检测到 GFP 荧光。在 25dpi 时，感染 pCCYVGFP 的黄瓜叶片表现出细胞扩散，而 pCCYVGFP 和 pCCYVGFP 主要存在于单个细胞中。进一步观察 30dpi、40dpi 和 50dpi 时 pCCYVGFP GFP 的表达情况，显示在系统叶片中 GFP 定位于韧皮部。
我们开发了 CCYV GFP 表达载体，这将有助于进一步研究 CCYV 在葫芦科植物中的运动。
