Cloning And Analysis Of Ta-hir3 And Ta-hir4 During The Interaction Between Wheat And Puccinia Triticina

Leaf rust is one of the most important diseases of wheat worldwide, and has also become one of the most important factors limiting good quality, high yield and stable production of wheat in China. Hypersensitive response is one of the most common resistant styles in wheat. The representative characteristic is that local necrosis around the infection point obviously occured during the host and pathogen interaction. The necrotic cells become dried, and the pathogens died for lacking nutrient. In the present study, wheat near isogenic line TcLr15 inoculated with diferent virulent leaf rust pathogen was used as the initial material, Ta-hir3 and Ta-hir4 cDNA and genomic DNA were amplified by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect the expression profiles of Ta-hir3 and Ta-hir4 in the compatible and incompatible interactions between wheat and leaf rust pathogen. Prokaryotic expression vectors with 2 hir genes were constructed, and transformed to E. coli competent cell. The fusion proteins produced by E. coli expression system were used to immune rabbit. The polyclonal antibodys from immuned rabbit were further used to detect the protein expression level in wheat leaves inoculated with different virulent leaf rust strains by Western blotting. Main contents were as followed:1. Total RNA of radicle, young leaves, young stems, immature seeds from wheat and flowers from different plant genus were extracted by NaAc/ethanol precipitation. The results showed that NaAc/ethanol precipitation is an inexpensive, flexible and rapid method for extracting total plant RNA. Result of the RT-PCR showed that expression profiles of five frequently used house-keeping genes (GAPDH, Actin, Rubisco, Ubiqutin, Tubulin)were different: GAPDH, Actin and Ubiqutin took on the same expression profiles. These three genes can be used as reference to detect the expression profile of related genes in wheat.2. Wheat isogenic line TcLr15 inoculated with avirulent strain 05-19-43â‘¡was used as the initial materials in the present study. Total RNA of wheat leaves at 24, 48 and 72 hours post inoculation (hpi) were mixed and used to synthesize first strand cDNA, and Ta-hir3 and Ta-hir4 cDNA were cloned by RT-PCR using the cDNA as template; And the gene DNA of Ta-hir3 and Ta-hir4 were also cloned with wheat genomic DNA as template. 3. Wheat cv. TcLr15 inoculated with avirulent strain 05-19-43â‘¡and virulent strain 05-5-137â‘¢w ere used as the initial materials, expression profile of Ta-hir3 and Ta-hir4 in wheat leaves at 0, 6, 12, 18, 24, 36, 48, 60, 72, 96, 120 hpi were detected by qRT-PCR.The result showed that Ta-hir3 and Ta-hir4 were strongly up-regulated when wheat was attacked by leaf rust pathogen, and showed extremely similar expression profile. Ta-hir3 transcript reached the expression peak at 48 and 36 hpi in the compatible and incompatible combination, respectively; Ta-hir4 started to increase at 18 hpi, and reached the peak at 48 hpi in the compatible combination. However, they likely started to increase at 6 hpi, and reached the peak at 36 hpi in the incompatible combination.4. After inserting Ta-hir3 and Ta-hir4 open reading frame into pGEM-T easy vecter, the recombinant plasmid Ta3-pGEM-T and Ta4-pGEM-T were digested with restriction enzyme EcoR I and Hind III, respectively. Digested fragment were directly inserted into the expression vector pET-30(+) digested with the same double enzymes. Two prokaryotic expression vectors with the target genes Ta3-pET-30a and Ta4-pET-30a were successfully constructed, and then were transformed into E.coli BL21(DE3). After optimization for the conditions of target protein producing, the molecular weights of the both fusion proteins were obtained with about 37 kDa.5. A sigle band appeared for the purified protein by SDS-PAGE after the fusion proteins were purified by Ni²⁺-His affinity columns. Antibody against Ta-HIR3 and Ta-HIR4 had been prepared by immuning rabbit with the purified protein. When valence of antibody reached to 1:76800, the antibody was isolated and used to dected the specificity by Western blotting. The result showed that the two proteins coresponding to Ta-HIR3 and Ta-HIR4 are existed in wheat.Total RNA of radicle, young leaves, young stems, immature seeds from wheat and flowers from different plant genus were extracted by NaAc/ethanol precipitation. The results showed that NaAc/ethanol precipitation is feasible, at the same time three house-keeping genes(GAPDH,Actin and Ubiqutin) can be used as reference to detect the expression profile of related genes in wheat. In this ressearch, we cloned Ta-hir3 and Ta-hir4 cDNA and DNA sequeces. Expression profile of Ta-hir3 and Ta-hir4 in wheat leaves were detected by qRT-PCR. The result showed that Ta-hir3å’ŒTa-hir4 expressed at the early time after attacked by leaf rust pathogen; The fusion proteins produced by prokaryotic expression system were used to immune rabbit, and the result of by Western-blotting showed that there existed the 2 proteins in wheat.