Molecular Analysis Of Three Stripe Rust Disease Resistance Related Genes In Wheat

Stripe rust, caused by Puccinia striiformis f.sp. tritici, is one of the most serious diseases in wheat worldwide. Breeding resistant varieties is an efficient way in controlling stripe rust. Therefore, it is very important to investigate molecular mechanism in the interaction of wheat and P. striiformis for breeding resistant varieties.In a previous study, an incompatible suppression subtractive hybridization (SSH) cDNA library of wheat leaves infected by Puccinia striiformis was constructed to identify genes that differentially expressed and involved in resistant response of wheat. In this study, three interesting genes from the SSH cDNA library were selected, and their molecular analyses were performed.A wheat 70-kDa heat shock proteins gene TaHSC70 was cloned using rapid amplification of cDNA ends technology. TaHSC70 is 2571bp in length. Sequence analysis showed that TaHSC70 included a complete 2073bp ORF and encoded a putative protein composed of 690 amino acids. The deduced amino acid sequence containes conserved domain and signature sites of 70-kDa heat shock proteins. It is highly homologous to chloroplast 70-kDa heat shock proteins in Oryza sativa, Ipomoea nil, Pennisetum glaucum, Cucumis sativus and Arabidopsis thaliana. By heat shock at 42?C, TaHSC70 transcripts keep in a steady state in normal wheat leaves, while increase about threefold in etiolated seedlings. In addition, in the interaction of wheat and stripe rust, the expression pattern revealed that TaHSC70 transcripts were up-regulated in incompatible group, whereas down-regulated after being up-regulated in the early stage in compatible group. It was speculated that TaHSC70 was involved in the interaction of stripe rust fungus and wheat by maintaining the physiological metabolism.Another wheat gene, 1563bp in length, was obtained by in silicon cloning and RT-PCR and designated TaAlaAT1. Sequence analysis showed that TaAlaAT1 included a complete 1440bp ORF and encoded a putative wheat alanine aminotransferase composed of 479 amino acids. The deduced amino acid sequence contains conserved aminotransferase signature and is highly homologous to alanine aminotransferase in Oryza sativa, Vitis vinifera, Glycine max, Arabidopsis thaliana and Medicago truncatula. The expression pattern of TaAlaAT1 at transcription level was investigated by semi-quantitative RT-PCR and real-time PCR, respectively, which revealed that TaAlaAT1 transcripts were up-regulated in incompatible interaction of wheat and stripe rust, whereas down-regulated in compatible interaction. It was speculated that TaAlaAT1 was involved in wheat defense response to P. striiformis.Previously, a new wheat hypersensitive induced reaction gene Ta-hir1 was cloned. Based on the full length cDNA sequence of Ta-hir1, its open reading frame was obtained by RT-PCR and the amplified fragments were cloned into pGEM^? T-easy vector. After restricted digestion with Hindâ…¢and BamHI, the ORF of Ta-hir1 was inserted into pET-32a vector and then the Ta-hir1-pET-32a recombinant was transferred to an E.coli strain Rosett(aDE3)plysS. Furthermore, a fusion protein, 47kDa, was successfully expressed by induction of IPTG and purified by His-Trap affinity columns. By way of injecting rabbit, the polyclonal antibodies were produced. The agar double immunodiffusion and ELISA test reveal that the antibody has good immune activity and high titer. Western blot analysis showed that the antibody reacted specifically to Ta-hir1 fusion protein and Ta-hir1 protein in wheat leaves.