棉花多抗品种中植棉KV-1抗病基因同源序列的克隆与分析

Isolation,Characterization and Phylogenetic Analysis of the Resistance Gene Analogues (RGAs) in Upland Cotton (Gossypium hirsutum L.) Zhongzhimian KV-1

许多抗病基因都具有核苷酸结合位点(NBS)和富亮氨酸重复区(LRR)。根据已知的NBS-LRR类抗病基因的保守序列,分别设计一对简并引物和一对特异引物,用以扩增棉花基因组中的抗病基因同源序列。获得一条大小约500bp的扩增片段,克隆测序后得到10条NBS-LRR类RGAs。推导的氨基酸均具有抗病基因的P-loop(kinase-1a)、kinase-2、kinase-3a及GLPL区。同源性比较发现,其中1条属于TIR-NBS-LRR类,其余9条属于non-TIR-NBS-LRR类。1条TIR-NBS-LRR类RGAs与已克隆的N、L6、M等抗病基因的同源性为51%～60%,9条non-TIR-NBS-LRR类RGAs与已克隆的RPS5、RPR1、Xa1等抗病基因的同源性为51%～60%。这些抗病基因同源序列(RGAs)可作为分子标记筛选棉花的抗病候选基因。

Most of known plant disease-resistance genes are featured with a nucleotide-binding site （NBS） and leucine-rich repeats （LRR）. A pair of degenerate primers and a pair of specific primer were designed based on the conserved NBS-LRR motifs among the known disease-resistance genes. The primers were used to amplify the disease-resistance gene analogues （RGAs） in upland cotton （Gos- sypium hirsuturn L. ）. A PCR product about 500 bp was obtained. After cloning and sequences of the DNA fragments, 10 NBS-LRR type RGAs were obtained. The deduced amino acid sequences of the DNA fragments contained the conserved motifs of NBS-LRR type RGAs, such as P-loop（kinase-la）, kinase-2,kinase-3a and GLPL domains. One of the RGAs is belonged to TIR-NBS-LRR type and the other nine are belonged to non-TIR-NBS-LRR type. When the TIR-NBS-LRR type RGA was compared with known disease-resistance gene such as N,L6,M, the percentage of amino acid homology ranged from 51% to 60% ,while the other nine non-TIR-NBS-LRR type RGAs had 51%-60% homologous compared with known resistance gene such as RPSS,RPR1,Xal and so on. These RGAs may be further used as molecular marker for screening of candidate disease-resistance gene in cotton.