桃基因组中R类抗病基因同源序列的克隆与序列分析

Cloning and sequencing of disease resistance gene analogues in peach (Prunus persica L. Batch)

许多抗病基因均具有核苷酸结合位点(NBS)和富含亮氨酸重复区(LRR).根据已知的NBS-LRR型抗病基因蛋白质的保守序列,设计简并引物,用以扩增桃基因组中的抗病基因同源序列.获得一条大小约500 bp的扩增片段,克隆测序后得到4个NBS-LRR类抗病基因同源片段(PNBS1、PNBS2、PNBS3、PNBS4).推导的氨基酸均具有P-loop(激酶1a)保守区和中间的激酶2a、激酶3a保守区,除PNBS4外,均具有3端疏水结构域.它们与已克隆的N、L6、PRS2等抗病基因在核苷酸水平上的同源性为21.1%～58.8%;在氨基酸水平上的同源性为18.8%～41.4%.这些抗病基因同源片段(RGA)可做为分子标记筛选桃的抗病候选基因.

Most of known plant disease-resistance genes are featured with a nucleotide-binding site (NBS) and leucine-rich repeats (LRR). The degenerate oligonucleotide primers were designed based on the conserved NBS motifs among the known disease-resistance genes. The primers were used to amplify the disease-resistance gene analogues (RGAs) in peach (Prunus persica L. Batch). A PCR product about 500 bp was obtained. After cloning and sequencing, 4 NBS-LRR type RGAs(PNBS1, PNBS2, PNBS3, PNBS4) in peach were detected. The deduced amino acid sequences of the DNA fragments contained the conserved motifs of NBS-LRR type RGAs, such as P-loop (Kinase 1a), Kinase 2a, kinase 3a and transmembrance domain (except for PNBS4). When the sequences of each RGA were compared with known resistance gene (N, L6, PRS2 etc.) sequences, the percentage of nucleotide identity ranged from 21.1%～58.8% and amino-acid identity ranged from 18.8%～41.4%. The RGAs may further be used as molecular marker for screening of candidate disease-resistance genes in peach.