Characterization And Functional Analysis Of Pathogen-induced MYB Transcription Factor Genes TaPIMP1 In Wheat

Zhou XianyaoDirected by:Prof. Zhang HuaiyuIn recent years, the production of wheat is seriously attacked by lots of diseases, as well as the atrocious weather while the environment change occurs. Especially, the diseases caused by Rhizotonia cerealis and Bipolaris sorokiniana, and the atrocious weather such as the drought and cold has affected the production of wheat in our country seriously. In this research, our intension is to the MYB transcription factor in wheat induced by pathogen. The character of expression, some properties of the transcription factor and the functions in transgenic tobacco and transgenic wheat have been studied.The study made the following results:1. The expression pattern of TaPIMPl was characterized. The results of Q-RT-PCR analysis indicated that the expression of TaPIMP1 could be enhanced in wheat leaves after inoculating Bipolaris sorokiniana and Rhizotonia cerealis. The response of this gene's expression is faster by B. sorokiniana than by R. cerealis. After inoculated by B. sorokiniana, the expression of TaPIMP1 could improve 3 times at 6h, and improve 11 times at 48h. However, in response to R. cerealis, the expression of TaPIMP1 arrive at the expression peak at 12h after inoculation, the expression could improve 3 times. In study the expression induced by treatment with defense phytohormones salicylic acid, jasmonic acid, ethylene and abscisic acid, the results indicated that the expression of TaPIMPl is more obvious induced by abscisic acid than by other phytohormones.2. TaPIMPl was domonstrated to be a nuclear localizing protein, and conditions of the expression in E.coli and the purification have been optimized. The TaPIMP1-GFP fusion protein vector has been constructed, and the vector was drive into onion epidermis cells by gene gun. After dark couture, the localization of fusion protein in cells was observed. The results of subcellular localization indicated that the TaPIMP1-GFP fusion protein was localized in the nucleus, whereas the control GFP was distributed throughout the cell, demostrated that TaPIMPl was targeted to the nucleus. In study the biochemistry character of TaPIMP1 protein in vitro, the vector of TaPIMP1-GST prokaryotic expression vector has been constructed, and then transfer into E.coli BL21 to do the prokaryotic expression. In study the expression in E.coli, the results indicated that when the inducing concentration of IPTG is 0.1 mM, the temperature is 16℃and the inducing time is 12h, the conditions of expression is appropreite, and the formation of inclusive body could be avoided.3. Defense fuctions of TaPIMPl to pathogens and abiotic stress in transgenic tobacco were confirmed. A series of TaPIMPl transgenic tobaccos were obtained by kanamycin filtering and PCR molecular detection. Furthermore, a series of over-expression TaPIMPl transgenic tobaccos were obtained by semi-quantitative RT-PCR. The results of Pseudomonas solanacearum inoculation in tobacco leaves indicated that the resistance to P. solanacearum in leaves of over-expression transgenic tobaccos is higher clearly than in leaves of wild type W38. The results of dehydration(20% PEG6000), salt(100mM NaCl) and oxidation treatment(300μM methyl viologen) to leaves indicated that the resistance to drought, salt and oxdation in leaves of over-expression TaPIMPl transgenic tobaccos is higher obviously. The results of drought and salt treatment to plants indicated that the resistance to drought and salt in plants of over-expression TaPIMP1 transgenic tobaccos is higher. It shows that plants of over-expression of TaPIMPl transgenic tobaccos have well effect of resistance to drought and salt. In analysis of physiological activity in transgenic lines, the total proteins were distilled. The results indicatied that the activity of phenylalanine ammonia-lyase and SOD is obviously higher than wild type.4. In molecular detection and disease resistant analysis to TaPIMPl transgenic wheat, the improved resistance to B. sorokiniana and R. cerealis in over-expression TaPIMPl transgenic wheat were confirmed. After PCR detection to transgenic wheat(Yangmail2) of TO and T1 generation, some stable TaPIMP1 transgenic wheat lines have been obtained. The results of R. cerealis and B. sorokiniana inoculation in wheat indicated that the resistant to R. cerealis and B. sorokiniana in transgenic wheat is higher than the receptor plant Yangmai12. The results demostrate that the TaPIMPl gene is positive regulate the wheat defense function.