An efficient virus-induced gene silencing (VIGS) system for functional genomics in Brassicas using a cabbage leaf curl v
- PDF / 1,773,974 Bytes
- 12 Pages / 595.276 x 790.866 pts Page_size
- 49 Downloads / 156 Views
ORIGINAL ARTICLE
An efficient virus‑induced gene silencing (VIGS) system for functional genomics in Brassicas using a cabbage leaf curl virus (CaLCuV)‑based vector Zhiliang Xiao1 · Miaomiao Xing1 · Xing Liu1 · Zhiyuan Fang1 · Limei Yang1 · Yangyong Zhang1 · Yong Wang1 · Mu Zhuang1 · Honghao Lv1 Received: 21 May 2020 / Accepted: 27 August 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Main conclusion CaLCuV-based VIGS effectively works in cabbage and contributes to efficient functional genomics research in Brassica crop species. Abstract 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 for Brassica oleracea until now. Here, tobacco rattle virus (TRV), pTYs and cabbage leaf curl virus (CaLCuV) gene-silencing vectors (PCVA/PCVB) were chosen to improve the VIGS system in cabbage using the phytoene desaturase (PDS) gene as an efficient visual indicator of VIGS. We successfully silenced the expression of PDS and 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, an Agrobacterium OD600 of 1.0, use of the vacuum osmosis method applied at the bud stage, and an incubation temperature of 22 °C. Using these parameters, we achieved a stable silencing efficiency of 65%. To further test the effectiveness of the system, we selected the Mg-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. rapa and B. 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. Keywords Brassicas · Cabbage · CaLCuV · Phytoene desaturase (PDS) · pTYs · VIGS system
Communicated by Dorothea Bartels. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00425-020-03454-7) contains supplementary material, which is available to authorized users. * Mu Zhuang [email protected] * Honghao Lv [email protected] 1
Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, 12# Zhongguancun Nandajie Street, Beijing 100081, China
Abbreviations CaLCuV Cabbage leaf curl virus ChlH Chelatase H subunit PDS Phytoene desaturase TRV Tobacco rattle virus VIGS Virus-induced gene silencing
Introduction Virus-induced gene silencing (VIGS), a pos
Data Loading...
