Jailani A Abdul Kader, Kumar Alok, Mandal Bikash, Sivasudha T, Roy Anirban
Advanced Centre for Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi, 110012 India.
Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, 620 024 Tamil Nadu India.
Virusdisease. 2016 Sep;27(3):277-286. doi: 10.1007/s13337-016-0326-8. Epub 2016 Jul 4.
Croton yellow vein mosaic virus (CYVMV, genus family ) is a proliferating begomovirus in the Indian sub-continent. The infectious constructs in binary vector was developed against the CYVMV genome and its associated betasatellite. Agroinoculation of the genomic construct of CYVMV produced leaf curl symptoms alone in three species of tobacco, , and . Co-inoculation of betasatellite enhanced the severity of the disease and reduced the incubation time. Based on the infectious clone, a replicon vector pCro, with only the ability to replicate inside the plant was developed. In pCro vector, CP and V2 ORFs from genome of CYVMV was deleted, which resulted localised replication of the molecule with no visible symptoms. Besides the partial CYVMV genome, pCro also has a cassette containing a double 35S promoter, multiple cloning sites and a NOS terminator to overexpress any foreign protein in plant. Episomal release of the replicon from the binary vector backbone after agroinoculation was detected by PCR. A GFP gene was cloned in pCro vector (pCro-GFP) and agroinoculated to resulted in localized expression of GFP at 5 dpi. The CYVMV replicon vector will be a useful tool for studying functional genomics, vaccine expression and gene silencing in plant.
巴豆黄脉花叶病毒（CYVMV， 属 科）是印度次大陆一种正在扩散的双生病毒。针对CYVMV基因组及其相关的β卫星构建了二元载体中的感染性构建体。用CYVMV基因组构建体进行农杆菌接种，仅在三种烟草（ 、 和 ）中产生了卷叶症状。β卫星的共接种增强了病害的严重程度并缩短了潜伏期。基于感染性克隆，开发了一种仅具有在植物体内复制能力的复制子载体pCro。在pCro载体中，删除了CYVMV基因组中的CP和V2开放阅读框，这导致该分子进行定位复制且无可见症状。除了部分CYVMV基因组外，pCro还具有一个包含双35S启动子、多克隆位点和NOS终止子的盒式结构，用于在植物中过表达任何外源蛋白。通过PCR检测农杆菌接种后复制子从二元载体骨架上的游离释放。将一个GFP基因克隆到pCro载体（pCro-GFP）中，并对 进行农杆菌接种，结果在接种后5天检测到GFP的定位表达。CYVMV复制子载体将成为研究植物功能基因组学、疫苗表达和基因沉默的有用工具。
