Cloning And Analysis Of TaHIR2 During The Interaction Between Wheat And Puccinia Triticina

Hypersensitive response (HR) is one of the most common resistant styles in plant. Hypersensitive induced response protein is a protein superfamily involved in plant HR. TaHIR2 had not been reported in wheat till now. In order to understand the existence of TaHIR2 in wheat and the relationship between TaHIR2 with wheat resistant to leaf rust pathogen, wheat near isogenic line TcLr15 inoculated with different virulent leaf rust pathogen was used as the initial materials, TaHIR2 full length cDNA and genomic DNA sequences were amplified by PCR. Quantitative real-time PCR (qRT-PCR) was used to detect the expression profiles of TaHIR2 in the compatible and incompatible interactions between wheat and leaf rust pathogen. Prokaryotic expression vector with TaHIR2 gene was successfully constructed, and transformed to E. coli competent cells. The fusion protein produced by E. coli expression system was used to immune rabbit and produce antibody. The expression profiles of TaHIR protein in wheat leaves inoculated with different virulent leaf rust strains were detected by Western blotting. The recombinant plasmids TaHIR2-pAHC-25 were constructed and transformed into embryogenic callus of wheat cv. TcLr15 and Thatcher by particle bombardment respectively. Main contents were as followed:1. Cloning and sequence analysis of wheat TaHIR2. Wheat near isogenic line TcLr15 and avirulent strain 05-19-43â‘¡were used as the initial material. Total RNA of wheat leaves at 24, 48 and 72 hours post inoculation (hpi) were mixed and used to synthesize first strand cDNA, and TaHIR2 cDNA and DNA were cloned by PCR using the cDNA and genomic DNA of TcLr15 as templates, respectively.2. Temporal and spatial expression profile of wheat TaHIR2. Wheat cv. TcLr15 inoculated with avirulent strain 05-19-43â‘¡and virulent strain 05-5-137â‘¢were used as the initial materials, temporal expression profile of TaHIR2 in wheat leaves at 0, 6, 12, 18, 24, 36, 48, 60, 72, 96, 120 hpi were detected by qRT-PCR. The results showed that TaHIR2 transcripts were up-regulated when wheat was attacked by leaf rust pathogen at 18 hpi, and reached the peak at 36 hpi both in the compatible and incompatible combination, then declined. But more transcripts were accumulated in incompatible interaction than compatible interaction at 24 and 36 hpi. Furthermore, four different tissues, including young roots, young stems, young leaves and mature seeds, from wheat cv. TcLr15 were used as the initial materials. The accumulation of TaHIR2 transcripts in the 4 tissues was detected by qRT-PCR, respectively. The results showed more TaHIR2 transcripts existed in young leaves.3. Expression profile of TaHIR2 protein in the wheat leaves inoculated with leaf rust at different time points. The prokaryotic expression vectors TaHIR2-pET-30a were successfully constructed after inserting open reading frame of TaHIR2 into pGEM-T easy vector, and then transformed into E.coli BL21 (DE3). After optimization for the producing conditions of target protein, the optimal IPTG concentration was 0.3 mM, the optimal inducing time was 7 h. The molecular weight of the fusion protein was about 37 kDa. The fusion protein expressed in inclusion body. A single band was detected by SDS-PAGE after the fusion protein was purified by Ni²⁺-His affinity columns. The purified protein was used to immune rabbit to produce polylclonal antibody. When potence of the antibody reached to 1:76800, the antibody was isolated and used to detect the specificity by Western blotting. The result showed that the protein corresponding to TaHIR2 was existed in wheat. The expression profiles of TaHIR2 protein were detected by Western blotting, and the results showed the accumulation of TaHIR2 increased steadily from 0 to 96 hpi in the incompatible combination, the peak occurred at 60 hpi, while the accumulation of TaHIR2 reached the peak at 96 hpi in the compatible combination.4. Construction of the plant expression vectors with TaHIR2 and obtaining of the regenerate wheat seedlings. The transgene vector TaHIR2-pAHC-25 was successfully constructed after inserting TaHIR2 open reading frame into plant expression vector pAHC-25, then transformed into the embryogenic callus of the wheat cv. TcLr15 and Thatcher by particle bombardment, the regenerate wheat seedlings were obtained after series of selective cultivation.Based on cloning of TaHIR2 cDNA and DNA sequences, series of analyses including qRT-PCR, western blotting and particle bombardment were carried out in this research. The results not only increase the understanding of wheat gene resources, but also make clear the functions of TaHIR2 in wheat, and supply new experimental supports for HR mechanism.