Development of plant virus vectors for genetic engeering in fruit frees

苹果球形潜伏病毒（Apple latent球形virus， ALSV）具有直径约25 nm的等距病毒颗粒，包含2种ssRNA （RNA1和RNA2）和3种衣壳蛋白。这种病毒被称为潜伏病毒，它感染大多数寄主植物时没有症状。本研究的目的是开发植物病毒载体，在植物中表达外源基因，并利用病毒载体在果树上建立基因工程体系。结果总结如下：(1) ALSV-RNA2载体的构建。成功构建了alsv - rna的感染性cDNA克隆，并将其修饰为病毒载体，实现了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，并将其应用于侵染植物的细胞间和长距离运动追踪。表达黄色和青色荧光蛋白（YFP和CFP）的ALSV也被构建并用于研究双重感染植物中相同但标记不同的病毒群体的相互作用。结果表明，ALSV-YFP和ALSV-CFP在侵染叶片中始终是分开分布的。ALSV载体还能表达植物中昆虫的抗真菌肽（滞育素）。(3)利用ALSV载体诱导转基因和植物内源基因的RNA沉默。表达绿色荧光蛋白（GFP）的ALSV感染有效地触发了转基因烟草植株中一种转基因基因（GFP基因）的病毒诱导沉默。我们还成功地用ALSV载体沉默了4种烟草（N.tabacum、N.benthamiana、N.occidentalis和N.glutinosa）和拟南芥的植物烯去饱和酶（PDS）、硫（SU）和增殖细胞核抗原（PCNA）基因。(4)表达拟南芥开花位点T （FT）基因的ALSV感染对植物开花的促进作用。表达FT的ALSV侵染后，4种烟草、矮牵牛花和拟南芥的开花均有明显促进作用。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