谷子毛状根诱导方法的建立与优化

Establishment and optimization of a high-efficient hairy-root system in foxtail millet(Setaria italica L.)

为建立一种快速鉴定谷子基因功能的技术体系,本研究通过比较谷子外植体、品种、乙酰丁香酮、菌液浓度和共培养时间对发根农杆菌K599介导的毛状根诱导效率的影响,发现发根农杆菌浓度在OD600为0.5、以谷子芽尖为外植体、在含有100μmol L–1乙酰丁香酮的毛状根诱导培养基上共培养3 d时,可使毛状根诱导率高达80.24%。将GFP基因进行毛状根遗传转化,通过GFP基因的PCR扩增和GFP荧光观察结果分析,发现谷子毛状根的转基因效率大于70%。利用该体系对谷子SiDVL1和SiDVL3的亚细胞定位和SiNHX2、SiCBL4和SiCBL7基因功能进行了分析和鉴定。结果表明SiDVL1和SiDVL3在谷子毛状根中的亚细胞定位与在烟草叶片中的一致;SiNHX2、SiCBL4和SiCBL7转基因谷子的存活率显著高于空载体转化谷子。说明本研究建立了一种高效快速鉴定谷子基因定位和功能的方法。

To establish a system for rapid identification of millet gene functions,in this study,we compared the effects of explants from different foxtail millet cultivars,the concentrations of acetyleugenone,bacterial solution,and co-culture time on the hairy root induction mediated by Agrobacteria rhizogenes strain K599.When the shoot tips were infected with bacterial solution 0.5 optical density(OD)and containing 100μmol L^(–1) acetyleugenone,and co-cultured for three days,the induction efficiency of hairy roots reached to 80.24%.Using this system,we analyzed the transformation efficiency of the hairy roots,the subcellular localization of SiDVL1and SiDVL3,and gene functions of SiNHX2,SiCBL4,and SiCBL7.The PCR results of GFP gene and GFP fluorescence microscopic observation indicated that the transformation efficiency of the hairy roots was over 70%.The subcellular localization of SiDVL1 and SiDVL3 was similar in millet hairy-root and tobacco epidermal cells.Simultaneously,the survival rates of SiNHX2,SiCBL4,and SiCBL7 transgenic millet were significantly higher than that of empty vector transformed millet.Therefore,our study established an efficient and rapid method to analyze the subcellular location and to identify the function of genes from foxtail millet.