Cloning, Expression And Functional Of Wrab17 Gene In The Process Of Wheat Infected By Puccinia Triticina

Wheat Leaf rust is caused by Puccinia triticina, affects the production of wheat(Triticum aestivum.L) seriously. It’s important to research the wheat resistance defense-related genes against Puccinia triticina for better studying the mechanism of wheat to Puccinia triticina resistance. Wrab17 belongs to wheat cold-responsive LEA/RAB-related COR protein. Our lab has engaged in the mechanism of wheat leaf rust infection for long time. We had established the SSH library and ESTs sequence analysis of the early wheat infected by Puccinia triticina in previous work. We found that Wrab17 showed significant up-regulation of expression in the incompatible combination of early vaccination. Thus, we presumed that the up-regulation of Wrab17 expression in the incompatible combination played an important role in the processs of the interaction between wheat and Puccinia triticina. HR(Hypersensitive reaction) is a common way of incompatible combinations against Puccinia triticina infection. The experiment aims to define whether Wrab17 participate HR in the process of wheat resistance to Puccinia triticina by using wheat variety ‘Lovirn10’(L10) with leaf rust races 260 to compose incompatible combinations. The main results are as follows:1.The CDS region of Wrab17 was cloned through the method of RT-PCR in wheat variety L10.2. Prokaryotic expression vectors for Wrab17 were constructed and transformed into E.coli BL21. By the way mentioned above, the prokaryotic expression of Wrab17 protein. Then we got the purified Wrab17 protein with His affinity purification. The rabbit anti-Wrab17 antibody was prepared and was detected by Western Blotting analysis, and polyclonal antibodywas obtained.3.We detected the expression variation of the m RNA level in Wrab17 in the wheat which is infected by Puccinia triticina at different time through the method of RT-PCR. In the early process of wheat to Puccinia triticina interaction, the expression of Wrab17 in incompatible combinations was significantly higher than the affinity combinations. We detected the expression variation of the protein level in Wrab17 in the wheat which was infected by Puccinia triticina at different time through the method of Western blot. And both expression had a similar trend. Therefor, Wrab17 involved in the defense of wheat to Puccinia triticina reaction.4.Subcellular localization vector of Wrab17 was constructed by tobacco injection, so Wrab17 expressed transiently in tobacco cells. We found the expression of Wrab17 in the cytoplasm and nucleus through fluorescence microscope.5.We constructed RNAi vector of Wrab17 and used transformation system of wheat mature embryos to transform wheat varieties 5389 and L10. Southern blot were initially obtained positive RNAi plants.6.Using RNAi to silent L10 Wrab17 gene, we found T1 positive plants which was infected by Puccinia triticina race 260 post 72 h induced HR area was reduced, and Puccinia triticina race 165 induced leaf rust increased. The result showed that Wrab17 gene plays an important role in wheat to Puccinia triticina resistance mechanisms.In summary, Wrab17 plays an important role in resistance to Puccinia triticina. This provides a better understanding of the role of Wrab17 in the immune response in plants.