果树病毒载体研究进展

doi:10.3864/j.issn.0578-1752.2014.06.008

摘要/Abstract

摘要： 植物病毒载体作为重要的研究工具被广泛运用于蛋白表达、基因沉默等研究，当前普遍使用的是以烟草花叶病毒载体等为代表的草本植物病毒载体，但其大多不能侵染果树，且稳定性较差，容易丢失插入的外源基因，因此该类载体无法满足果树等多年生植物研究的需要。近年来新兴的果树病毒载体可解决这些难题，为此作者对果树病毒载体的研究进展和发展方向进行综述。当前国内外取得的研究成果主要包括：（1）通过掌握柑橘衰退病毒、柑橘叶斑病毒、李痘病毒、苹果潜隐球形病毒、葡萄病毒A和葡萄卷叶伴随病毒等果树病毒的传播途径、寄主范围、致病力分化、基因功能和表达策略等特性，采用体外转录或农杆菌介导的方式获得了上述果树病毒的全长侵染性克隆。在此基础上，通过在病毒外壳蛋白基因与其邻近的上游基因之间插入外源基因（包括荧光蛋白基因和β-葡萄糖醛酸酶基因等报告基因），并用该病毒外壳蛋白基因的启动子或其他异源启动子驱动外源基因表达的方式，将上述6种果树病毒的全长侵染性克隆改造成为病毒载体；（2）运用果树病毒载体明确了柑橘衰退病毒、柑橘叶斑病毒、李痘病毒、苹果潜隐球形病毒和葡萄卷叶伴随病毒在植株中的分布、移动规律，及其在细胞中的定位。探寻了柑橘衰退病毒在大翼来檬上产生茎陷点症状的原因，以及交叉保护防治柑橘衰退病的主要机理。果树病毒载体还被作为病毒诱导的基因沉默载体用于基因功能和防病研究；（3）通过选用本地已经存在，且无虫传能力的弱毒株，以及对控制病毒致病和媒介传播能力的基因进行敲除、突变可以解决果树病毒载体研发过程中所遇到的安全风险。由于有些果树病毒仅分布于植株的韧皮部，因此限制了其作为病毒载体在植株中表达外源基因的范围，但由这类果树病毒构建的病毒载体稳定性极高，并且通过添加分泌信号肽基因等方式可以扩大表达产物在植株中的分布和作用范围，因此其在果树病毒研究方面仍然具有很高的应用价值。此外，采用不同病毒来源的异源启动子代替同源重复区来驱动外源基因的表达，可以进一步提高果树病毒载体的稳定性。

关键词: 果树 , 病毒载体 , 柑橘衰退病毒

Abstract: Virus-based vectors are commonplace tools for the production of proteins or induction of RNA silencing in plants. But even if the existing vectors from herbaceous plant viruses could infect fruit trees, the time for systemic infection and analysis of the expressed genes in trees generally exceeds the stability of known virus-based vectors. Now this problem has been solved by using virus-based vectors from fruit trees. Progress in studies on virus-based vectors from fruit trees was summarized. The results obtained in recent years are as following: (1) The transmission, host range, differentiation of pathogenicity, genome organization and regulation of gene expression of Citrus tristeza virus (CTV), Citrus leaf blotch virus (CLBV), Apple latent spherical virus (ALSV), Plum pox virus (PPV), Grapevine virus A (GVA) and Grapevine leafroll-associated virus (GLRaV) have been elucidated. Construction of Agrobacterium-mediated infectious cDNA clones or infectious RNA transcripts derived from the full-length cDNA clones of these fruit crops viruses. And then, a foreign open reading frame (ORF) such as green fluorescence protein (GFP) gene, β-glucuronidase gene (GUS) was inserted between coat protein (CP) gene and the adjacent gene as a reporter. The expression of the foreign gene was driven by a duplicated native CP subgenomic (sg) RNA controller element (CE) or an introduced heterologous CE of other virus. (2) Virus-based vectors from fruit trees have been used to elucidate cell-to-cell as well as long-distance movement, spatial separation, localization within the host cells, the processes of stem pitting induced by CTV in Citrus macrophylla and the mechanism of CTV superinfection exclusion. These vectors also can be used as virus induced gene silencing (VIGS) vectors to elucidate gene function. Furthermore, CTV-RNAi vector has been demonstrated to silence the endogenous genes of Diaphorina citri and Candidatus Liberibacter asiaticus. (3) From the environmental safety standpoint, the virus used as the basis of the vector should not cause diseases of plants and not be transmitted by natural vector. Thus, one should choose the non-vectored mild strains. A second approach is to mutate the viral vector, eliminate the determinants needed for pathogenicity and vector-mediated spread. Some viruses of fruit trees are limited to phloem-associated cells. Although the vectors are not the appropriate vectors for expression of genes in other tissues, phloem-limited viruses have resulted in the most stable vectors. Furthermore, this limitation can be reduced somewhat by producing proteins with secretion signal peptides to export the protein out of the cell into the intercellular space. Finally, the stability of virus-based vectors from fruit trees could be improved by using promoters from different tobamoviruses instead of repeated promoters.

Key words: fruit tree , virus-based vector , Citrus tristeza virus

周彦. 果树病毒载体研究进展[J]. 中国农业科学, 2014, 47(6): 1119-1127.

ZHOU Yan. Progresses in Study of Virus-Based Vectors of Fruit Trees[J]. Scientia Agricultura Sinica, 2014, 47(6): 1119-1127.

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