数字农科院2.0

An Efficient Virus-Induced Gene Silencing (Vigs) System For Functional Genomics In Brassicas Using A Cabbage Leaf Curl Virus (Calcuv)-Based Vector

文献类型： 外文期刊

作者： Xiao, ZL; Xing, MM; Liu, X; Fang, ZY; Yang, LM; Zhang, YY; Wang, Y; Zhuang, M; Lv, HH

作者机构：

关键词： Brassicas; Cabbage; Calcuv; Phytoene Desaturase (Pds); Ptys; Vigs System

期刊名称： PLANTA

ISSN： 0032-0935

年卷期： 2020 年 252 卷 3 期

页码：

收录情况： JCR（2021版）

摘要： Main conclusion CaLCuV-based VIGS effectively works in cabbage and contributes to efficient functional genomics research in Brassica crop species. Virus-induced gene silencing (VIGS), a posttranscriptional gene silencing method, is an effective technique for analysing the functions of genes in plants. However, no VIGS vectors have been available forBrassica oleraceauntil now. Here,tobacco rattle virus(TRV), pTYs andcabbage leaf curl virus(CaLCuV) gene-silencing vectors (PCVA/PCVB) were chosen to improve the VIGS system in cabbage using thephytoene desaturase(PDS) gene as an efficient visual indicator of VIGS. We successfully silenced the expression ofPDSand observed photobleaching phenomena in cabbage in response to pTYs and CaLCuV, with the latter being more easy to operate and less expensive. The parameters potentially affecting the silencing efficiency of VIGS by CaLCuV in cabbage, including the targeting fragment strategy, inoculation method and incubation temperature, were then compared. The optimized CaLCuV-based VIGS system involves the following: an approximately 500 bp insert sequence, anAgrobacteriumOD(600)of 1.0, use of the vacuum osmosis method applied at the bud stage, and an incubation temperature of 22 degrees C. Using these parameters, we achieved a stable silencing efficiency of 65%. To further test the effectiveness of the system, we selected theMg-chelatase H subunit(ChlH) gene in cabbage and knocked down its expression, and we observed yellow leaves, as expected. We successfully applied the CaLCuV-based VIGS system to two other representative Brassica crop species,B. rapaandB. nigra, and thus expanded the application scope of this system. Our VIGS system described here will contribute to efficient functional genomics research in Brassica crop species.

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