Cloning And Characterization Of The Promoters Of Powdery Mildew Resistance Gene Stpk-V In Haynaldia Villosa And Its Orthologous Gene In Wheat

Wheat powdery mildew disease, which is caused by Blumeria graminis f. sp. tritici, is one of the most serious wheat diseases in China and many other countries of the world. Pm21, which is from Haynaldia villosa and located on the short arm of chromosome6V showed high level and wide spectrum resistance to powdery mildew of wheat. Pm21are now widely used in wheat breeding for powdery mildew resistance. Stpk-V, the Bgt upregulated gene screened by genechip, was cloned as a key member of the Pm21locus. It can improve the disease resistance after transformed into the susceptible wheat Yangmai158. Although6AS,6BS and6DS of common wheat all contain the orthologous genes of Stpk-V, the common wheat is still susceptible to powdery mildew. To study the resistant mechanism of Stpk-V, the promoter of Stpk-V and its orthologous genes were cloned and characterized, and the function of the orthologous gene Ta-D-Stpk was evaluated by making it overexpression in the susceptible Yangmai158.1. The Ta-D-Stpk was transformed into the susceptible Yangmail58based on the constructed vector pAHC25:Ta-D-Stpk by biolistic transformation strategy, and the resistance function of Ta-D-Stpk to powdery mildew was evaluated.2. The promoters of the orthologous genes of Stpk-V was cloned by using the primers designed according to promoter of the Stpk-V gene. The promoter of Stpk-V was designated as pStpk-V, and the promoters from the common wheat was designated as pTa-A-Stpk, pTa-B-Stpk,pTa-D-Stpk. The promoters were placed upstream of the GFP gene to form the recombinant vector, named as pSGFP-pStpk-V, pSGFP-pTa-A-Stpk, pSGFP-pTa-B-Stpk, pSGFP-pTa-D-Stpk, and the transient expression system was used to detect the constitutive activity of these promoters. At the same time, the promoters were placed upstream of the GUS gene to to form the recombinant vector, named as pAHC25-pBI220-pStpk-V, pAHC25-pBI220-pTa-A-Stpk, pAHC25-pBI220-pTa-B-STPK, and pAHC25-pBI220-pta-D-Stpk, amd the transient expression system was used to detect the induced actitivity of the promoters. By transient expression analysis, the promoting activity of the pStpk-V and other three promoter from the common wheat was compared.The results were as follows:1. In the leaved transformed with pSGFP plasmids, a large number of GFP-expressing cells could be observed, however, in the leaves transformed with pSGFP-pStpk-V, pSGFP-pTa-A-Stpk, pSGFP-pTa-B-Stpk and pSGFP-pTa-D-Stpk, newly no GFP-expressing cells were detected. Therefore, it was suggested that the pStpk-V, pTa-A-Stpk, pTa-B-Stpk and pTa-D-Stpk have no constitutive promoting activity.2. In the leaves transformed with pAHC25-pBI220-pStpk-V, pAHC25-pBI220-pTa-A-Stpk and pAHC25-pBI220-pTa-pTa-Stpk, the GUS-expressed cells could be detected in the Bgt inoculated leaves, however, there was no GUS-expressed cells in the Bgt uninoculated leaves. It was also found that there were more GUS-expressed cells in the pAHC25-pBI220-pStpk-V transformed leaved than the leaved transformed with other three recombinant plasmids. It was suggected that the promoting activity of these Stpk genes was induced by Bgt, and the activity of the pStpk-V was higher than those promoters from the common wheat after the Bgt inoculation.3. Six To Ta-D-Stpk transgenic plants were obtained by gene-gun mediated gene transformation approach. The function of the Ta-D-Stpk was evaluated using the T1generation plants, and the result indicated that over-expression of Ta-D-Stpk could not enhance the resistance of the Yangmai158to the powdery mildew disease.