Development of plant virus vectors for genetic engeering in fruit frees

苹果潜伏球状病毒(ALSV)具有等长的病毒颗粒，直径约25 nm，含有两种单链RNA(RNA1和RNA2)和三种衣壳蛋白。这种病毒是一种潜伏病毒，会毫无症状地感染大多数寄主植物。本研究的目的是开发在植物中表达外源基因的植物病毒载体，并利用该病毒载体建立果树基因工程体系。主要结果如下：(1)ALSV-RNA2载体的构建：成功构建了ALSV-RNAs的感染性克隆，并将其改造成病毒载体，用于外源基因在植物中的稳定表达。(2)利用ALSV载体在植物中表达外源基因。构建了表达绿色荧光蛋白(GFP)的ALSV载体，并用于病毒在受感染植物中的细胞间和长距离移动的示踪。表达黄色和青色荧光蛋白(YFP和CFP)的ALSV也有…更多的被构建和用于研究在双重感染的植物中相同但不同标记的病毒群体的相互作用。结果表明，ALSV-YFP和ALSV-CFP总是分别分布在病叶中。利用ALSV载体在植物中诱导转基因和内源基因的RNA沉默。表达GFP的ALSV在转基因烟草中有效地引发病毒诱导的转基因基因(GFP基因)沉默。我们还成功地通过ALSV载体沉默了四个烟草种(烟草、本氏烟草、西方烟草和粘性烟草)和拟南芥中的植烯脱饱和酶(PDS)、硫磺(SU)和增殖细胞核抗原(PCNA)基因。(4)表达拟南芥开花基因T(FT)的ALSV促进了植株的开花。较少

Apple latent spherical virus (ALSV) has isometric virus particles, c. 25 nm in diameter, and contains two ssRNA species (RNA1 and RNA2) and three capsid proteins. The virus is what is called a latent virus that infects most host plants symptomlessly. The aims of the research are the development of plant virus vectors for expressing foreign genes in plants and the establishment of the genetic engineering system in fruit trees by the virus vectos. The results were summarized as follows.(1)Construction of the ALSV-RNA2 vectors.Infectious cDNA clones of ALSV-RNAs were successfully constructed and modified into viral vectors for stable expression of foreign genes in plants.(2)Expression of foreign genes in plants using ALSV vectors.The ALSV vector which expresses green fluorescence protein (GFP) has been constructed and used for the tracing of cell-to-cell and long distance movement of the virus in infected plants. ALSV expressing yellow and cyan fluorescent proteins (YFP and CFP) have also … More been constructed and used investigate the interaction of populations of identical, but differently tagged viruses in doubly infected plants. The results indicated that ALSV-YFP and ALSV-CFP were always distributed separately in infected leaves. The ALSV vector could also express an antifungal peptide (diapausin) from an insect in plants.(3)Use of ALSV vectors for inducing RNA silencing of a transgene and endogenous genes in plants.ALSV expressing GFP infection efficiently triggered virus-induced gene silencing of a transgene (GFP gene) in transgenic tobacco plants. We also successfully silenced the phytoene desaturase (PDS), sulfur (SU), and proliferating cell nuclear antigen (PCNA) genes in four Nicotiana species (N.tabacum, N.benthamiana, N.occidentalis, and N.glutinosa) and Arabidopsis thaliana plants by ALSV vectors.(4)Promotion of flowering in plants Infected with ALSV expressing flowering locus T (FT) gene of Arabidopsis thaliana.The infection of ALSV expressing FT resulted in the strong promotion of flowering in four Nicotiana species, petunia, and A.thaliana. Less