Cloning And Characterization Of Wheat Resistance-related Genes From TcLr35

Cloning of plant resistance gene is greatly helpful to crop resistance breeding and the insight of resistance mechanism. The wheat genome is extremely large containing approximately 16,000Mb of DNA, 40 times larger than rice, and contains 80% repetitive DNA sequences. That makes it seemingly laborious to use a map-based approach to clone the genes if they are distributed randomly throughout the genome. Cloning of the homologous fragments, locating and linkage analysis with the cloned ones provided a new idea for wheat resistance gene isolation. In this study, the primers were designed based on the conserved domains of the cloned resistance genes before. The homology-based cloning for the candidate resistance genes from wheat was carried out combining with rapid amplification cDNA end and thermal asymmetric interlaced PCR. In addition, wheat pathogenesis-related protein genes were screened by RT-PCR and RACE. The main results as follows:(1) The primers were designed according to the amino acid conserved regions of the reported plant disease resistance genes encoding proteins containing the nucleotide-binding sites (NBS) and leucine-rich repeats (LRR). Four coding sequences named as PS13-1, PS13-2, S2A2, and LRR1 were obtained respectively from the RNA of TcLr35 carrying the Lr35 gene conferring resistance against wheat leaf rust by reverse transcription-polymerase chain reaction (RT-PCR). Three RGAs were isolated containing the conserved motifs of NB-ARC such as P-loop, kinase2, kinase3a and the transmembrance domain. LRR1 contained three LRR repeat units, and had high homology with Xa21.(2) The gene specific primers were designed based on the sequence of the resistance gene homologues S2A2, then the complete sequence of wheat resistance-related gene S2A2 were obtained through the 5'RACE and 3'RACE methods. The length of S2A2 was 2693bp including a 1887bp complete open reading frame encoding S2A2 protein of 628 amino acids, a 230bp 5'untranslated region (5'UTR), a 533bp 3'untranslted region (3'UTR) and 21bp poly (A) tails. The initiation codon and the stop codon were found in 233bp and 2117bp locating respectively. The deduced amino acids of S2A2 protein consisted of a CCdomain, a nucleotide binding site (NBS) domain, a leucine-rich repeats (LRR) domain, which were the conserved domains of plant resistance genes. It was suggested that the S2A2 gene 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 DQ205351. The S2A2 gene appeared not to be induced by Puccinia triticina and was a constitutive gene with low abundance in the genome by semi-quantitative RT-PCR.(3) Two fragments with 51 lbp and 614bp in length were amplified from TcLr35 by thermal asymmetric interlaced PCR (TAIL-PCR). The fragments overlapped with the known RGA1 sequences with 293bp and 509bp respectively. A 915bp sequence was obtained through extension RGA1 by 208bp at 3'end and lOObp at 5'end. BLASTn analysis, it showed that 93 % identical to wheat leaf rust resistance gene Lr21 and 90% identical to the contig which contained LrlO.(4) Two pairs of primer were designed based on the pathogenesis-related protein 1 and fl (1, 3;1, 4) beta glucanase, two full length cDNA sequence were obtained from TcLr35 induced by the puccinia triticina by RT-PCR and RACE. A full length wheat pathogenesis related protein 1 gene named PR12 was 756bp in length, including a 495bp complete open reading frame encoding 164 amino acids, a 147bp 5' untranslated region (5' UTR), a 155bp 3' untranslated region (3' UTR) and 21-bp poly (A) tails. The deduced amino acids contained SCP conserved domains which was related to plant defense systems. The deduced amino acid sequence showed close homology to PR-1 like proteins, which have been isolated from many plants. Southern blot indicated that the wheat genome contained single copy of PR12 gene;A full length wheat 13 (1, 3;1, 4) beta glucanase gene named PR34 was 1340bp in length, which included one complete open reading frame encoding 303 amino acids, a 323bp 3' untranslated region (3' UTR) and 29-bp poly (A) tails. The initiation codon and stop codon were found in 77bp locus and 988bp locus respectively. The deduced amino acid sequence showed close homology to beta glucanase proteins which have been isolated from wheat, barley, and many other plants. The deduced amino acids contained Glyco_hydro₁7 conserved domains which was related to beta glucanase. Southern blot indicated that the TcLr35 genome contained several copies of PR34 gene.