Construction Of RANi Expression Vector Of Dehydrin Gene WZY2from Wheat And Its Genetic Transformation

Drought is an important environmental factor that restricts agricultural production. Theglobal arid and semiarid region accounts for about36%of the total land area and43%of thecultivated land area. Our country arid and semiarid region area accounts for about half of thenational land area, mainly distributed in the northwest area. The wheat is one of main foodcrop, and drought stress influences on plant growth and cuts down the crop production by10%~20%. Therefore, the study of drought resistance of wheat has important meaning tosolve the world food problem.Dehydrin is a kind of hydrophilic protein with thermal-stability which accumulated inthe late period of embryogenesis. It can be induced by environment stresses, such as cold,high temperature, drought and salt stress. It can reduce the drought injury on plant byprotecting the stability of cell membrane and acting as a molecular partner in the protection ofbiological metabolism. The wzy2gene is cloned from zhengyin No.1by our laboratory. It is anew dehydrin member, but in the role of drought on it is still not clear.The RNA interference technique can regulate gene expression efficiently and speciallyby silencing or knocking out the gene at the post-transcriptional level. Now it has been widelyused in the research of gene function. So by the principle of RNA interference andhomological analysis of wzy2, we chose185bp cDNA on wzy2between553bp and737bp asthe interference fragment and constructed the middle vector named pKANNIBAL-FR whichcontained reverse repeated sequence by ligation the interference fragment to thepKANNIBAL vector. Then by inserting the reverse repeated sequence into pAHC25vectorwhich is an efficient plant expression vector and contains the Ubi promoter, the RNAiexpression vector pAHC-wzy2-Ri was constructed. Then pAHC-wzy2-Ri vector wastransformed into wheat1500embryos of zhengyi NO.1by biolistic particle and103regenerated plants were obtained. The regeneration rate is6.87%.16of the103regeneratedplans were Bar-positive identified by PCR by using the specific primer of Bar and theBar-positive ratio is1.01%.3of16Bar-positive plant were positive identified by PCR byusing the specific primer of the reverse repeat sequence and the positive ratio is0.2%. Get theseeds of T0transgenic plants and grow part of them. We get15plants of T1progency.11of T1 generation transgenic plants were Bar-positive identified by PCR, and2of them theinterference fragment were positive. This study laid a foundation for further research of thefunction of wzy2gene.