水培条件下病毒诱导棉花基因沉默体系的建立及优化

以国欣棉3号为材料,以棉花GhCLA1为指示基因,探讨了生长温度、重悬液浓度、注射时间、品种等对水培棉花pTRV介导的VIGS沉默效率的影响。在24℃条件下,出苗后3~5 d内注射能得到较高沉默效率,重悬液浓度对沉默效率没有影响;同时以注射pTRV-GFP作为空白对照可以消除插入片段对植株生长的影响,减小对照误差;水培与土培方式相比能更快更早出现沉默表型,缩短试验周期,并能诱导不同品种棉花材料GhCLA1基因沉默;利用水培棉花TRV-VIGS体系,成功抑制了棉花GhCTR1基因的表达,与对照株相比,抑制后的棉花植株出现矮化表型,说明水培棉花TRV-VIGS体系建立在棉花研究中的广谱利用性。

Establishment and Optimisation of Virus-Induced Gene Silencing in System Hydroponic Cotton

This experiment using GhCLA1 as a marker gene and cotton variety Guoxinmian 3 plants as material was conducted to explore effects of temperature, syringe-infiltrated concentrations and time, cultivation patterns, and cotton varieties on efficiency of tobacco rattle virus (TRV)-induced gene silencing (VIGS) under hydroponic condition. The results showed that higher silencing efficiency was induced by syringe-infiltrated time at 3 to 5 days after emergence and optimum growth temperature at 24 ºC under hydroponic condition, but syringe-infiltrated concentrations could not affect VIGS silence efficiency. Moreover, pTRV-GFP as null fragment could alleviate the adverse effect of inserted fragment for plant growth. Silencing phenotype could be visible earlier in hydroponics culture than in soil culture, and the experimental period was significantly shortened under hydroponic condition. In addition, GhCLA1 could be silenced in all tested varieties (lines) under hydroponic condition. Cotton plants with silenced GhCTR1 were severely dwarfed, which indicated TRV-VIGS system can be applied widely in hydroponic cotton.