J Integr Plant Biol. ›› 2003, Vol. 45 ›› Issue (3): 340-345.

• Research Articles • Previous Articles     Next Articles

Isolation of Resistance Gene Analogs from Wheat Based on Conserved Domains of Resistance Genes

QIN Gen-Ji, CHEN Pei-Du, GU Hong-Ya, FENG Yi-Gao and NIU Ji-Shan   

  • Published:2003-03-01

Abstract:

Two pairs of degenerate primers were designed based on nucleotide-binding site (NBS) and serine/threonine kinase domain. PCR was performed with the primers and cDNA from the Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL. Amplified products were cloned and sequenced. Nine clones with NBS and one with serine/threonine kinase domain were obtained. The NBS clones were classified to six groups according to their nucleotide sequence identities (90% or higher). These resistance gene analogs (RGAs) all have open reading frames (ORF), and their amino acid sequences show high similarity to Yr10 in wheat, Mla1 and Mla6 in barley, RPS2 in Arabidopsis and other resistance (R) genes with conserved motifs. They were preliminarily mapped on the chromosomes of homoeologous groups 1, 2 and 5 of common wheat by nulli-tetrasomic analysis. The 5′-end sequence of an RGA N5 was obtained by 5′-RACE PCR. It encodes six leucine zipper (LZ) and has high sequence similarity to RPS2. Key words: resistance gene analogs; nucleotide-binding site; PCR; Triticum aestivum-Haynaldia villosa translocation line

根据 R 基因保守区分离小麦R  基因类似序列
秦跟基 陈佩度  顾红雅 冯 高 牛吉山

(1. 南京农业大学农业部作物细胞遗传重点实验室,南京210095;
2. 北京大学生命科学学院,蛋白质工程及基因工程国家重点实验室,北京100871)

摘要: 用根据核苷酸结合位点(nucleotide binding site, NBS)和丝氨酸/苏氨酸蛋白质激酶域设计的2对简并性引物,以小麦-簇毛麦6VS/6AL易位系的cDNA为模板进行PCR扩增.扩增产物克隆到pGEM-T载体中,经测序,共获得具有NBS结构域特征的片段克隆9个和具有丝氨酸/苏氨酸蛋白质激酶域特征的片段的克隆1个.将克隆之间核苷酸序列同源性高于90%的克隆归为一类,把9个NBS片段分为6类.这6类抗病基因类似序列(resistance gene analogs, RGA)均具有阅读框,并与已克隆的小麦抗条锈病基因Yr10、大麦抗白粉病基因Mla1和Mla6、拟南芥的抗病基因RPS2以及其他一些抗病基因在NBS保守区内具有高度的同源性.用小麦中国春缺体-四体初步将它们分别定位于小麦第一、第二和第五部分同源群上.进一步用5′-RACE技术获得RGA N5的5′-端,发现其编码产物的N端还具有6个亮氨酸拉链(leucine zipper, LZ),与RPS2的N-端有较高同源性.

关键词: R 基因类似片段;核苷酸结合位点;:PCR;小麦-簇毛麦6VS/6AL易位系

通讯作者。Tel: 025-4396026; Fax: 025-4395344;E-mail: pdchen @ njau.edu.cn.

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