Cloning Of A CDNA Fragment Of Resistance-related Gene And The Candidate For The Stripe Rust Resistance Gene Yr10 From Wheat

AbstractStripe rust (yellow rust), caused by Puccinia striiforms f. sp. tritici West., is one of the most important diseases on wheat throughout the world. Now we haven't found the cloning of wheat rust disease resistance gene. Wheat stripe rust disease resistance gene YrlO confer resistance to all the races in China. So we used wheat cultivar Moro(containing YrlO gene) and NIL of YrlO as materials to clone the resistance-related cDNA fragments and candidate gene for YrlO.Using the total RNA extracted from seedlings of wheat cultivar Moro (YrlO) inoculated with the avirulent isolate CY27 and the virulent 75078 of Puccinia striiformis West, respectively, we optimized silver-stained mRNA differential display RT-PCR to get a plentiful and high-resolution silver PAGE. Combining three anchored primer T11MN (M is degenerate of A, G, C; N is A, G or C) with 40 10-mer arbitrary primer, differences on mRNA level were analyzed among compatible interaction, incompatible interaction and not inoculated control. Some primer combinations amplified plentiful bands, while others combinations amplified only a few bands.A total of 146 polymorphic bands were reamplified from the samples collected 24, 48, 72, 96h after inoculation. A cDNA fragment DD-1 was cloned with T-easy vector after reverse Northern blotting of 78 differential displayed bands from the samples of 24 and 48h and sequencing showed that it was 316bp (GenBank accession number AYO15491). It has no obvious homologous sequences in GenBank.Northern dot blot hybridization analysis using probe of DD-1 as the probe uncovered that DD-1 was induced by both isolates, but the transcript abundance was less in the compatible interaction than in the incompatible interaction. Seventy-two hours after inoculation, the transcript abundance of DD-1 had no obviously change in the incompatible interaction though it decreased in the compatible interaction.Degenerated primers were designed according to the conserved NBS domain of R genes. Amplification was done with Touch Down PCR using the cDNAs astemplates reverse transcribed from YrlO NILs mRNAs. A 771bp PCR product appeared in the resistant Fir/0/6 X Avocet S but not in the susceptible background parent Avocet S. It has a high homology (99%) with YrlO gene(accession in Dec. 2000) in both nucleotide and amino acid sequences.With primers designed according to the sequence of the DNA fragment, an about 2.0kb fragment was amplified from the genomic DNA of YrlO/6 X Avocet S. It showed that this gene has an about 1.0kb intron.To get the 5'-terminal and the 3'-terminal fragments, a pair of primers was synthesized according to 12-30 of the 771bp cDNA and 1-18 of YrlO mRNA and a pair of primers according to 752-771 of 771bp cDNA and 3611-3630 of YrlO mRNA. A 636bp fragment at the 5'-terminal and an 1118bp fragment at the 3'-terminal were amplified by PCR with the primers from the cDNA of YrlO/6X Avocet S. The same fragments were also amplified from the genomic DNA.The full sequence of the mRNA was gained by combining the 3 cDNA fragments. The full mRNA with 2475 nucleotides has an ORF containing 824 amino acids. The deduced protein primary structure has LZ-NBS-LRR domain. Southern blotting using the genomic DNA digested with EcoR I and BamH I and the DIG-labeled 1118bp probe showed that the obtained gene is of single copy.The primers using to amplify 1118bp fragment at the 3'-terminal binds to YrlO gene specifically. The 1118bp fragment is a good molecular marker of wheat stripe rust disease resistance gene YrlO.