The Expression Profile Analysis Of Six UniGenes Induced By Wheat Stripe Rust And Isolation Of TaLHY And TaNIT Gene

Wheat stripe rust, caused by Puccinia striiformis f.sp.tritic, is the major disease in China. Especially in west regions of our country and due to suitable conditions, the disease recently turns to be most serious there. The reason can be concluded as fast variation of pathogen, lack of resistance genes and single resistant inheritance basis of wheat variety and complicated resistant mechanism between pathogen and plant interaction. The molecular mechanism is still unclear so as to restrict the breeding of new resistant variety. Thereby, while constantly finding out new resistant genes and fast breeding of new wheat variety with durable resistance researches should be focused on molecular mechanism of pathogen-host recognition and plant resistance and cloning resistance genes and resistant-related genes, all of which are crucial for fundamentally solving threats from wheat stripe rust.From previous constructed SSH cDNA library of rust-resistant variety named CN19 induced by Puccinia striiformis Chinese race CY32, we obtained a large number of differentially expressed ESTs. In this study, we hope to probe into the molecular mechanism of disease resistance induced by stripe rust and explore resistance genes related to stripe rust resistance, thus,six UniGenes were selected to carry out the expression profile analysis and two genes were selected to obtain the full length cDNA using RACE and in silico cloning combined with RT-PCR. As follows:1 Semi-quantitative RT-PCR analysis revealed the expression profile of the six UniGenes from the SSH-cDNA library, respectively functional annotations were VTC2, SAMDC, SHMT, galactose-binding lectin, JIP and LHY protein.â‘ tissue-specific expression analysis showed that six UniGenes can be able in the jointing stage of root, stem, leaf and ear, and the seedling root and leaf tissues, special in the ear or the root with the relatively high expression level;â‘¡pathogens induced expression results showed that six UniGenes were significant difference between CN19 infected by stripe rust and powdery mildew pathogen. Six UniGenes accumulated to high expression level within 24-48 houres when CN19 inoculated by CY32. While, only the galactose-binding lectin, VTC2 and JIP gene began to express after disseminated the powdery mildew fungus on CN19 48hours later and peaked at 96 hours;â‘¢hormone induced expression analysis showed that all of the six UniGenes expression induced by ethylene (Et) and showed different pattern responded to salicylic acid (SA) treatment and abscisic acid (ABA). The rusluts suggested that the incompatible interaction between CN19 and CY32 was regulated via Et/JA singaling pathway. SA and ABA can regulate the expression of some defense response genes;â‘£JIP, galactose-binding lectin gene and SHMT also involved in cold treatment, dehydration and high salinity stress responses.2 A MYB transcript factor TaLHY was cloned from SSH-cDNA library by using RACE-PCR. The full cDNA length of TaLHY was 3085bp including a 1947bp ORF which induced a 648 AA polypeptide with molecular weight of 70.3kDa and pI 6.34. It contained a typical MYB-DNA binding domain and a conservative SH[AL]QKY[RF] in C-terminal of the domain and was highly homologous with other LHY family members. Subcellular localization of TaLHY was on the cell nucleus and it participated in the transcript regulations and signal transductions, which demonstrated that it maybe a new MYB gene. The expressing level of TaLHY was up-regulated after 48 hours infected by stipe rust race CY32 and up-regulated in 1 hour and 4 hours after treated with Et, which proved that TaLHY was involved in the resistance response of wheat defending the rust infection and maybe play the role via the JA/Et pathway.3 A wheat nitrilase gene TaNIT was cloned by using in silico cloning combined with RT-PCR. The full cDNA length of TaNIT was 1457bp and its ORF was 1023bp,.It encodes a 340 amino acid protein with calculated molecular weight of 36.3 kDa and pI of 5.70. TaNIT has a typical nitrilase family domain and the conservative Glu-Lys-Cys catalytic triad. The prediction of subcellular localization of TaNIT was a soluble cytoplasmic protein loosely combined with the plasma membrane. TaNIT as a lyase involved in energy metabolism and the plant immune response.