Cloning And Characteristic Analysis Of Genes In The Interaction Between Wheat And Stripe Rust

Stripe rust caused by Puccinia striiformis f. sp. tritici is a kind of serious disease in wheat worldwide. Cloning resistant-related genesand breeding resistant varieties is an efficient way in controlling stripe rust. In our laboratory, a suppression subtractive hybridization (SSH) cDNA library of shuiyuan11 wheat leaves induced by Puccinia striiformis Chinese race CY31 was constructed, and obtained large subtractive ESTs. 3 genes were selected and their elementary characteration was analyzed.In this study, the full length cDNA were obtained by screening cDNA library using PCR method. However, the result was not perfect sometimes, and then RACE and in silico cloning was used to obtain the full length cDNA. RNA was extracted from wheat leaves inoculated with P. striiformis after 0h, 12h, 18h, 24h, 48h, 72h and 120h. CDNA prepared from the mixed RNA was used as the template of Real-time RT-PCR for analyzing genes'expression mode. Prodaryotic expression and VIGS system was designed. As follows:1 Peroxiredoxin of wheat was cloned, and named TaPrx. Gene specific primers were designed based known EST. The full length cDNA of TaPrx gene was 688bp, and its ORF was 489bp,It encoded a 162 amino acid protein with calculated molecular weight of 17.36 kDa and isoelectric point of 5.32. The deduced protein included one conserved one cysteine, however, the signal peptide and transmembrane helices were not found. The prediction of subcellular localization of TaPrx gene was cytoplasmic with 94% probability. 6-160aa was PRX5 family conserved motif. The molecular weight of TaPrx fusion protein is 38 kDa.Western blot showed that the antiserum had a good specificity with fusion protein. Real-time RT-PCR indicated that the expression of TaPrx gene was induced by Puccinia striiformis in wheat, and the highest expression occurred at 24h and 18h after inoculation in compatible and incompatible interaction respectively, consistent with ROS expression mode. The TaPrx gene may participate in eliminating and regulating ROS in wheat challenged by Puccinia striiformi.2 Engulfment and cell motility protein(ELMO)of wheat (named TaELMO) was cloned by screening cDNA library and in silico cloning. The full length of TaELMO gene was 1155bp, and its ORF was 810bp. It encodes a 269 amino acid protein with calculated molecular weight of 30.657 kDa and isoelectric point of 5.51. The signal peptide and transmembrane helices were not found. The prediction of subcellular localization of TaELMO gene was cytoplasmic with 91% probability. Real-time RT-PCR showed a lessened mode of the expression of TaELMO gene in wheat induced by Puccinia striiformi.3 The homology gene of Zea mays lethal leaf-spot 1 of wheat (named TaLls1) was cloned by RACE and in silico cloning. The full length of TaLls1 gene was 1927bp, and its ORF was 1605bp. It encodes a 534 amino acid protein with calculated molecular weight of 59.178 kDa and isoelectric point of 8.46.There was a chloroplast targeting peptide and one transmembrane helices in the sequence, and conserved Rieske[2Fe-2S] motif and non-heme mononuclear iron-binding sites also were included. Real-time RT-PCR indicated that the expression of TaPrx gene was induced by Puccinia striiformis in wheat.The VIGS system of TaLls1 gene was designed successfully.