以陆地棉CLA1基因为标记基因,利用烟草脆裂病毒(tobacco rattle virus, TRV)载体建立基于病毒介导的棉花基因沉默体系(virus-induced gene silencing, VIGS)。病毒RNA2的RT-PCR分析证明,棉花子叶接种TRV病毒后,该病毒可高效扩散到受体的根、茎、叶等器官。利用TRV-VIGS体系同时诱导34份不同来源棉花材料CLA1基因沉默,尽管不同材料间的抑制程度有差异,但均可有效抑制 CLA1基因的表达,说明该体系在棉花研究中的广谱利用性。GhMAPKKK 基因受黄萎病菌诱导表达,接种后96 h 表达量达到高峰。利用 TRV-VIGS 体系,成功抑制了棉花GhMAPKKK 基因的表达,与对照株相比,抑制后的棉花植株接种黄萎病菌后更易感病,说明 GhMAPKKK 参与了棉花对黄萎病菌的抗性反应。具有广谱性、灵敏性和高通量等特点的棉花 TRV-VIGS 体系建立将加速棉花功能基因组研究进程。%Using upland cotton GhCLA1 as marker gene, tobacco rattle virus induced genes silencing (TRV-VIGS) was estab-lished in cotton. A gene fragment of TRV subgenomic RNA2 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) in root, stem and leaf of cotton, demonstrating the virus can spread into various organs. The TRV induced silencing of CLA1 gene was further tested in 34 different cotton varieties (lines) originated from several ecological regions of China. The re-sults showed that CLA1 could be silenced in all tested varieties (lines), though the levels of silencing showed a little difference, implicating wide application perspective of TRV-VIGS system in cotton. GhMAPKKK, a mitogen-activated protein kinase (MAPK) kinase kinase gene of cotton, was up-regulated in cotton at 96 hours post inoculation by Verticillium dahliae. Silencing GhMAPKKK in G. barbadense cv. Hai 7124, a cotton variety with high resistance to V. dahlia exhibited reduced resistance to V. dahliae infection, suggesting that GhMAPKKK participated in cotton resistance signaling pathway to V. dahliae. With the wide adaption without genotype selection, sensitivity and high throughput, the TVR-VIGS system will significantly promote functional gene analysis in cotton.
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