Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India.
CSIR-Central Institute of Medicinal and Aromatic Plants Research Centre, Bangalore, India.
Plant Biotechnol J. 2015 Dec;13(9):1287-99. doi: 10.1111/pbi.12347. Epub 2015 Mar 24.
Withania somnifera (L.) Dunal is an important Indian medicinal plant that produces withanolides, which are triterpenoid steroidal lactones having diverse biological activities. To enable fast and efficient functional characterization of genes in this slow-growing and difficult-to-transform plant, a virus-induced gene silencing (VIGS) was established by silencing phytoene desaturase (PDS) and squalene synthase (SQS). VIGS of the gene encoding SQS, which provides precursors for triterpenoids, resulted in significant reduction of squalene and withanolides, demonstrating its application in studying withanolides biosynthesis in W. somnifera leaves. A comprehensive analysis of gene expression and sterol pathway intermediates in WsSQS-vigs plants revealed transcriptional modulation with positive feedback regulation of mevalonate pathway genes, and negative feed-forward regulation of downstream sterol pathway genes including DWF1 (delta-24-sterol reductase) and CYP710A1 (C-22-sterol desaturase), resulting in significant reduction of sitosterol, campesterol and stigmasterol. However, there was little effect of SQS silencing on cholesterol, indicating the contribution of sitosterol, campesterol and stigmasterol, but not of cholesterol, towards withanolides formation. Branch-point oxidosqualene synthases in WsSQS-vigs plants exhibited differential regulation with reduced CAS (cycloartenol synthase) and cycloartenol, and induced BAS (β-amyrin synthase) and β-amyrin. Moreover, SQS silencing also led to the down-regulation of brassinosteroid-6-oxidase-2 (BR6OX2), pathogenesis-related (PR) and nonexpressor of PR (NPR) genes, resulting in reduced tolerance to bacterial and fungal infection as well as to insect feeding. Taken together, SQS silencing negatively regulated sterol and defence-related genes leading to reduced phytosterols, withanolides and biotic stress tolerance, thus implicating the application of VIGS for functional analysis of genes related to withanolides formation in W. somnifera leaves.
睡茄（Withania somnifera（L.）Dunal）是一种重要的印度药用植物，可产生具有多种生物活性的三萜甾体类内酯物。为了实现对这种生长缓慢且转化困难的植物中基因的快速有效的功能鉴定，我们通过沉默类胡萝卜素脱饱和酶（PDS）和鲨烯合酶（SQS）建立了病毒诱导的基因沉默（VIGS）。沉默编码鲨烯合酶的基因（为三萜提供前体）导致鲨烯和甾体化合物显著减少，证明其在研究睡茄叶中甾体化合物生物合成中的应用。对 WsSQS-vigs 植物中的基因表达和甾醇途径中间产物进行全面分析，揭示了转录调节，其中甲羟戊酸途径基因呈正反馈调节，下游甾醇途径基因呈负前馈调节，包括 DWF1（Δ24-甾醇还原酶）和 CYP710A1（C-22-甾醇去饱和酶），导致豆甾醇、菜油甾醇和谷甾醇显著减少。然而，SQS 沉默对胆固醇的影响很小，表明甾体化合物的形成主要依赖于豆甾醇、菜油甾醇和谷甾醇，而不是胆固醇。在 WsSQS-vigs 植物中，分支点角鲨烯合酶表现出不同的调节，导致 CAS（环阿屯醇合酶）和环阿屯醇减少，BAS（β-香树脂醇合酶）和 β-香树脂醇诱导。此外，SQS 沉默还导致油菜素内酯 6-氧化酶 2（BR6OX2）、病程相关（PR）和非表达 PR（NPR）基因下调，导致对细菌和真菌感染以及昆虫取食的耐受性降低。总之，SQS 沉默负调控甾醇和防御相关基因，导致植物甾醇、甾体化合物和生物胁迫耐受性降低，从而暗示 VIGS 在睡茄叶中与甾体化合物形成相关基因的功能分析中的应用。
