Optimization Of Transient Expression System In Wheat Leaves And VIGS Analysis Of Three RGAS

Transient expression technology has been widely used in various plants,it is an efficient method for rapid characterization of gene function.Compared with the stable transformation method,transient expression technology does not need to integrate of transgenes into plant genomes and can study gene expression or function in short time.In transient expression system,we often use gene gun,Agrobacterium,protoplast and viral vector to mediate gene transformation.The gene gun-mediated transient expression system is widely used for its applicability to diverse receptor tissues and host types.In wheat leaf transient expression system,there are still some problems exist such as low efficiency,poor stability and uneven bombardment.Using GFP and GUS as reporter genes,this study optimized the transient expression system in wheat leaf through four parameters including treatment of wheat leaves,plasmids DNA concentration per shot,plasmids coating method and the distance between stopping screen and target cells.The optimized system improved the efficiency of transient expression by more than one fold compared to the previous study.In studying the interaction between wheat leaf cells and powdery mildew,the number of interacting cells was increased by 35.8%-44.6%using these optimized parameters.We used this optimized system to silence the recessive resistance gene Mlo in susceptible wheat leaves and further confirmed the effectiveness of our optimized system.In our laboratory,we isolated a BAC from an einkorn wheat accession,the BAC carried three resistance gene analogs RGA2185,RGA3 and RGA11.Expression analysis indicated that the expression levels of these three genes were up-regulated after induced by powdery mildew.This study preliminarily analyzed the function of the three genes using VIGS technology.The result showed that all the three genes were silenced in different levels after infected with virus for 7 days,but only plants infected with RGA3 showed susceptibility,indicating that RGA3 was resistant to powdery mildew.