| Leaf rust disease, caused by the fungal pathogen Puccinia triticina, is one of the most severe diseases of common wheat (Triticum aestivum L.) all over the world. Breeding and application of resistant cultivars are the most economical and environmentally safe approach to reduce the damage caused by wheat leaf rust disease. The resistance gene Lr19 which was transferred into wheat from Agropyron elongatum cultivars has expressed resistance to most pathotypes of puccinia triticina in most regions of the world. As a resistance gene to wheat leaf rust, Lr19 would have high potential in application at home and abroad.In this study, the degenerated primers were designed according to the nucleotide binding site conserved domains of the cloned plant disease resistance (R) genes. The methods of homology-based cloning and RACE were used to obtain the full length sequence of the disease resistance homology gene in the near isogenic lines TcLr19. The main results are as follows:1. Two coding sequences named as S11A11 and CIN14 were obtained respectively from the RNA of TcLr19 carrying the Lr19 conferring resistance against wheat leaf rust by reverse transcription-polymerase chain reaction (RT-PCR) using the primers according to the amino acid conserved regions of the reported plant disease resistance genes encoding proteins containing the nucleotide binding site (NBS). The deduced amino acids of S11A11 and CIN14 protein contained the conserved motifs of NB-ARC such as P-loop, kinase2, kinase3a and the transmembrance domain.2. The gene specific primers were designed based on the sequence of the resistance homologue gene S11A11, then the complete sequence of S11A11 was obtained through the 5′RACE and 3′RACE methods. The full length of S11A11 was 2923bp including a 2637bp complete open reading frame encoding S11A11 protein of 878 amino acids, a 161bp 5′untranslated terminal region (5′UTR), a 100bp 3′untranslated terminal region (3′UTR) and 25bp polyA tails. The deduced amino acids of S11A11 protein consisted of a CC domain, a nucleotide binding site (NBS) domain, and a leucine-rich repeats (LRR) domain, which were the conserved domains of plant resistance genes. It suggested that the S11A11 gene should be a member of a class CC-NBS-LRR type resistance gene. The gene has been submitted to the GenBank database, with the accession number of GU356592.3. The gene specific primers were designed based on the sequence of the resistance homologue gene CIN14, then the complete sequence of CIN14 was obtained through the 5′RACE and 3′RACE methods. The full length of CIN14 was 2987bp including a 2643bp complete open reading frame encoding CIN14 protein of 880 amino acids, a 155bp 5′untranslated terminal region (5′UTR), a 138bp 3′untranslated terminal region (3′UTR) and 35bp polyA tails. The deduced amino acids of CIN14 protein consisted of a CC domain, a nucleotide binding site (NBS) domain, and a leucine-rich repeats (LRR) domain, which were the conserved domains of plant resistance genes. It suggested that the CIN14 gene should be a member of a class CC-NBS-LRR type resistance gene. The gene has been submitted to the GenBank database, with the accession number of GU356591.4. TcLr19, which was uninoculated and inoculated at different times (0h, 6h, 12h, 18h, 24h, 36h, 48h, 60h, 72h, 96h) by using the method of semi-quantitative RT-PCR, was analyzed. The results showed that the S11A11 and CIN14 genes had the same expression level before and within 96 hours after the TcLr19 was inoculated, and suggested that S11A11 and CIN14 genes be constitutive genes with low abundance in the wheat leaf tissue during the samping time period. |
