基因枪介导的小麦叶片瞬间表达体系的优化及应用

麦类作物的叶片单细胞瞬间表达是一种高效而快速的基因功能验证方法,尤其适用于麦类作物与白粉病菌的互作系统,但由于影响参数较多、参检基因表达频率低而尚未广泛应用。本研究使用绿色荧光蛋白(GFP)基因作为报告基因,对影响表达效率的多个转化参数进行优化,筛选出表达频率最高的参数组,即小麦叶片采用固体保鲜培养基固定,钨粉用量为每枪300μg,质粒DNA用量为每枪750ng,可裂膜氦压为1 350psi,可裂膜与受体膜距离6mm,受体与阻挡网距离6cm,与先前报道的方法相比表达频率有显著提高。应用上述参数在感病小麦品种离体叶片中过量表达一个已知具有抗白粉病功能的簇毛麦SGT1基因,结果表明该基因瞬间表达使互作细胞吸器指数降低,表现为对白粉病菌侵入和吸器形成具有抑制作用,在一定程度上增强了表达细胞对白粉病菌的抗性,使目标基因的抗性功能再次得到印证。本研究还进一步运用该优化体系开展瞬间表达介导的基因沉默(TIGS)实验,将携带抗白粉病基因Pm21的抗病小麦品种南农9918和感病小麦品种扬麦158中的内源SGT1和RAR1分别或同时沉默,结果显示南农9918的叶片表皮与白粉菌互作细胞的吸器指数增高,在一定程度上降低了表达细胞对白粉菌的抗性。而扬麦158中互作细胞的吸器指数较对照变化不显著。说明这两个基因均分别参与了Pm21介导的抗白粉病信号途径,且二者共同作用与白粉病抗性关系更加密切。

Establishment and Application of an Optimized Transient Expression System of Wheat Leaves Using Particle Bombardment

The transient expression system using wheat leaf epidermal cells is a highly efficient method for rapid characterization of gene function, especially in the interaction between wheat and Blumeria graminis f. sp. tritici (Bgt). But because of the influence of many parameters and low expression efficiency of the target gene, the system was not yet widely used. GFP, the green fluorescence protein gene was used as the reporter gene to optimize the parameters of biolistic transformation protocol by counting the GFP expressed wheat leaf cell after transient expression by bombardment. Optimal parameters with the highest transient expression frequency were obtained through repeated experiment and data statistics. Solid and retain freshness media was chosen to fasten the wheat leaves before and after the particle bombardment. The amount of tungsten powder was 300 ng each bombardment. The concentration of plasmid DNA was 750 ng each bombardment. He pressure of the rupture disc was 1350 psi and the distance between the rupture disc and the microcarrier (A) was 6 mm, the distance between the macrocarrier and the transgenic acceptor (B) was 6 cm. Compared with our previous report, the new system had outstanding advantages and obvious effects. Using the optimal parameters, the SGT1 gene isolated from Haynaldia villosa, which had been proved involving in Bgt resistance, when transiently overexpressed in leaf epidermal cells of susceptible wheat variety, could decrease the haustorium index, partly inhibit the penetration of conidiospores and formation of haustoria, and to some extent increase the resistance of cells to powdery mildew. So the resistant function of Hv SGT1 had been confirmed again. On the contrary, SGT1 and RAR1 was transiently silenced by harpin RNA mediated method respectively or synchronously in the resistant wheat variety Nannong 9918, which lead to an increase in the haustorium index of the interacting cells, and increase the resistance of the cells to Bgt. But same transiently silencing of SGT1 and RAR1 in susceptible wheat variety Yangmai158 resulted in no significantly change of the haustorium index. The results implied that either one of SGT1 and RAR1 was involved in the Pm21 mediated Bgt resistance pathway, and joint action of SGT1 and RAR1 showed closer relation to Bgt resistance.