| Isolation of plant resistance gene is greatly helpful to crop resistance breeding and the insight of resistance mechanism. No functional resistance gene has been separated from soybean at present. Sequences of cloned resistance genes from a wide range of plant reveal significant similarities in amino acid sequence. In this study, we cloned a resistance-related gene from soybean with homology-based candidate gene method used the degenerate oligonucleotide primers which were designed with the conserved structures of cloned resistance genes. Our study became the basis of the soybean functional resistance gene cloning and improving the resistance of soybean through transgene technique. The main experimental results were as follows:1 Degenerate oligonucleotide primers were designed according to the Arabidopsis RPS2 and tobacco N and flax L6, and about 100 clones were isolated from the RNA of resistance soybean cultivar Suinong10, resistant to phytophthora sojae, and two open-reading NBS resistance gene analogs were finally characterized and named as RNEAU-1 and RNEAU-2. The length of the two analogs were both 513bp which code 171 amino acids. The 171 amino acids of the two analogs consisted of the conserved structures of nucleotide binding site (NBS) domain which includes P-loop, Kinase-2a and Kinase 3a and conserved hydrophobic domain(HD). Homology of sequence and structure of the two analogs with resistance genes (fragment) of other plants showed they were resistance gene homologues in soybean. The two analogs has been submitted to the GenBank database, and the accession number are AY 182243 AY 182244 respectively.2 Based on the sequence of one of resistance gene homologues RNEAU-1, we designed gene specific primers, and got the complete sequence of soybean resistance-related gene through the 5' RACE and 3' RACE methods. The resistance-related gene was named SR1. The length of SR1 was 3574bp which included a 3411-bp complete open reading frame encoding SR1 protein of 1137 amino acids, a 72-bp 5'non-translated region(5'NTR), a 68-bp3'non-translated region(3'NTR)and 20-bp poly(A) tails. The initiation codon and stop codon were found in 73bp locus and 3484bp locus respectively. The deduced 1137-amino acids SR1 protein consisted of Toll/Interluekin receptor (TIR) domain, a nucleotide binding site (NBS) domain, a leucine-rich repeats(LRR) domain, a hydrophobic domain and a conserved domain2, which were the conserved domains of plant resistance genes. TheSR1-related sequences were presented as 2-4 members in soybean genome according to the result of southern blotting. The SR1 gene was constitutive gene with low abundance in soybean genome. The transcripts of SR1 gene were detected in roots, stem and leaves of soybean. The gene appeared not to be induced by phytophthora sojae inoculation and salicylic acid treatment. Due to these features the SR1 gene was a member of class I TIR-NBS-LRR type resistance gene. The product of SR1 was resistance-related protein which was similar to tobacco N in structure. The gene has been submitted to the GenBank database, and the accession number is AY193892.3 Designed specific primers according to the sequence of SR1, a PCR reaction was done with the genome DNA of Suinong10 as template. A 3972bp fragment was acquired finally which was named SR2.The SR2 gene consisted of 5 exons and 4 introns according to the sequence alignment. The junction sequences of exons and introns accorded to the classical cut and assembly sequence GT/AG The length of 5 exons were 478bp , 1106bp, 290bp, 793 and 779bp respectively, and that of the 4 introns were 195bp, 156bp, 87bp and 88bp respectively. Except of the 4 introns, the other sequence of SR2gene shared a homology of 100% with the SRJ gene.4 Designed specific primers according to the sequence of SR1, PCR reactions were done with the genome DNA of five other resistance soybean cultivars Dongnong163, Dongnong9674, Dongnong43, Dongnong93046 and Suinong8 and a sensitive soybean cul... |
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