TRV 病毒诱导大豆基因沉默体系优化及应用.

Virus-induced gene silencing（VIGS）technology has been widely used in plant gene function research. The efficiency of gene silencing in soybean mediated by tobacco rattle virus（TRV）-based vector system remains to be clarified. The seamless cloning technique was used to construct a TRV-VIGS system to explore the silencing efficiency in soybean target genes in different tissues with different inoculation methods, providing a basis for gene function studies in soybean. Taken phytoene desaturase（GmPDS）and ubiquitin ligase（GmATL3）as target genes, pTRV1 and the recombinant vectors solution were inoculated into soybean（Glycine max）plants Zhonghuang13 by three methods: injection, agroinoculation and a combination of injection plus agroinoculation. The silencing phenotypes were observed at 28 d after inoculation, and the relative expressions of the genes in the roots and leaves were quantified by RT-qPCR to determine the silencing effectiveness of the different methods. Silencing of GmPDS resulted in yellowing and chlorosis on the edges and inside of the leaves, while chlorosis spots and fold chlorosis phenotypes in the leaves surface occurred. The results of RT-qPCR showed that silencing efficiency of GmPDS of all three inoculation methods was close to 100%. The silencing efficiency of GmATL3 by the injection was 80%-95% in the leaves and 40%-60% in the roots; the silencing efficiency of the root inoculation and injection plus root inoculation was 70%-90% in roots and 15%-50% in leaves. Different inoculation methods caused different levels of silencing phenotypes and silencing efficiencies for different endogenous genes. The silencing efficiency of an inoculation method on different tissues was different, and the highest silencing efficiency on the leaves and roots were injection method and injection plus agroinoculation, respectively. [ABSTRACT FROM AUTHOR]