Cloning And Pathogen-induced Expression Analysis Of TaHsp90.4 And TaMA3 In Wheat Defense Response Against Strip Rust

Wheat stripe rust (Puccinia striiformis f.sp.tritic), the main diseases of wheat, is particularly serious in western China. The resistance of new wheat varieties with resistant genes to Puccinia striiformis f.sp.tritic is prone to failure because this disease has a variety of races with variability and frequency. Currently, there is only a few Puccinia striiformis f.sp.tritic resistant genes can be utilized. Therefore, finding more Puccinia striiformis f.sp.tritic resistant genes as well as addressing the molecular mechanism of interactions between host and Puccinia striiformis f.sp.tritic would be the foundation of genetics and breeding of wheat to disease. Wheat variety 19 (CN19) containing stripe rust resistance gene Yr41, In this paper, we based on pre-constructed CN19 SSH cDNA library with CN19 which induced by Puccinia striiformis f.sp.tritic race CY32, chosen two EST sequences (unigen32 and unigen42) cloned two resistant genes and researching expression pattern induced by stripe rust CY32, This study was to explore the mechanism of resistance defense response by stripe rust and host interaction,which is the scientific basis for the identification and cloning Yr41 candidate genes and regulatory genes, The main results obtained are as follows:1. A HSP90 family gene has been cloned, the gene is different from the wheat published in TaHsp90.1, TaHsp90.2, and TaHsp90.3 gene by amino acid sequence alignment and secondary and tertiary structure prediction, tentatively named TaHsp90.4. TaHsp90.4, with a total length 2536bp, contains an open reading frame of 2424bp which encoding 807 amino acids. This protein showed high similarity with Hsp90 of Urartu wheat, wheat and barley,99%,94% and 96%, respectively. Secondary and tertiary structure prediction indicate that it has a classic conserved domains for Hsp90:an ATP-binding domain in N terminal, a middle (M) domain for customer protein recognition and C terminal for binding to Hsp90 dimer. In addition, a specific KEDL sequence for endoplasmic reticulum protein Hsp90 was also existed and a casein kinase â…¡ phosphorylation sites charge region between N and M terminal.2.Cloning of a typical four tandem repeat copy MA3 domain protein gene, tentatively named TaMA3.TaMA3, with a total length 2040bp, contains an open reading frame of 1999bp which encoding 666 amino acids. This protein showed high similarity with Brachypodium, Aegilopstauschii and Urartu wheat, consistency as 91%,97% and 98%, respectively. Close evolutionary distance was found in Aegilopstauschii and Urartu wheat. TaMA3 has a typical repeat domain of MA3 with four tandem repeat copies, which was similar to Arabidopsis topoisomerase subsets, consistency as 39%.3. This study used quantitative PCR to research expression characterization of TaHSP90.4 at wheat seedling, jointing and heading stage by the stripe rust induced, the valuable information was provided for research and elucidating with the function of TaHSP90.4 gene and stripe rust resistance of wheat inter-relationships.Relative expression of TaHSP90.4 was highest in the leaf, and significantly increased followed by heat shock for lh. In resistant varieties CN19 with Puccinia striiformis f.sp.tritic induced, relative expression of TaHSP90.4 was inhibited and then increased, and finally decreased with obvious expressed peak. In susceptible varieties MY11, the expressed peak of TaHSP90.4 was not obvious, suggesting that it might participate in immune response in susceptible varieties and the activation of protein R as well as the identification process of signal transduction for R protein to Puccinia striiformis f.sp.tritic and protein damage repair.4. Relative expression of TaMA3 was higher in leaves and young inflorescences. In CN19 and MY11 of with Puccinia striiformis f.sp.tritic induced, relative expression of TaMA3 increased first, then recovered with obvious expression peak in al three different growth stages, suggesting that it might participate in immune regulation to wheat pathogens. Expression peak of TaMA3 in CN19 was significantly higher than MY11, suggesting that it may be playing a more important role in resistant varieties.