Cloning And Expression Of E3 Gene In The Process Of Wheat Infected By Puccinia Triticina

This research is based on the formal studies. Yan et al. had established a SSH library of the reaction between the wheat and leaf rust, from which the TaE3 EST was selected. And this EST has a high expression level in the wheat leaves at 16 hours after the inoculation of leaf rust 366. The formal studies have indicated that in the stress of high or low temperature, some of the proteins in plants would have fault enfolding, whose accumulation would result to deceases. For instance, the AtCHIP protein which has the activity of E3 is repairing and digesting the abnormal proteins by operating the expression of Hsp70 and HSP90, and then reduce the negative effects of the environmental stress. In this research, the wheat TcLr19 and leaf rust 366 were used as the materials, and the TaE3 gene was cloned by the method of RT-PCR. Then, the qRT-PCR and Western Blotting were used to identify the expression of the TaE3 in the early stage after the leaf rust infects the wheat in both mRNA and protein levels. After that, the eukaryotic expression vector was obtained and then transformed into the Arabidopsis theliana. Hence, the TaE3 overexprssion plants were obtained. The mainly results are as follows:1: The TaE3 gene was cloned by the method of RT-PCR from the wheat seedlings'leaves.2: The prokaryotic express vector of TaE3 gene was constructed, and a large quantity of the fusion protein was obtained. The anti-rabbit serums of the TaE3 multi-clones were used to identify the specificity between TaE3 and the anti-serums.3: The varietal expression of TaE3 in both mRNA and protein level at different hour's post the inoculation of leaf rust was verified by qRT-PCR and Western Blotting, respectively. The results shows that, this gene has rarely varied before 8 h, however, it reached its peak expression level at 16 h. In the protein level, TaE3 start to increase at 8 h, and attained a high level at 16 h, after that, it reduced at 24 h, and the expression level at 48 h approximated the control. And this could afford a primary evidence of the participation of TaE3 gene in the Hypersensitive Response when the leaf rust infects wheat leaves.4: The eukaryotic expression vector pCAMBIA1300-35S:: TaE3-eYFP was obtained and then transformed into the Arabidopsis theliana. Hence, the TaE3 overexprssion plants were obtained.All the results above made a foundation for the further study of the TaE3's function and the mechanism in the reaction between the wheat and leaf rust.