SON-PCR扩增抗禾谷孢囊线虫基因LRR区及序列分析

根据与抗禾谷孢囊线虫(Heterodera avenae)基因Cre3的NBS(nucleotide binding site)编码区高度同源的Rccn4 序列设计3条3'端嵌套引物,采用SON-PCR(single oligonucleotide nested PCR)方法从含有源于易变山羊草(Aegilops varibilis)的抗禾谷孢囊线虫基因的小麦(Triticum aestivum)-易变山羊草染色体小片段易位系E-10中获得了长度为1264 bp的Rccn-L(GenBank登录号为DQ124933)，它将Rccn4 3'端延伸了1209 bp,编码区长1026 bp，含一个不完整的开放阅读框,一个终止密码子，没有起始密码子和内含子结构。其编码一个342个氨基酸残基的蛋白质，含有NBS(nucleotide binding site)-LRR(leucine-rich repeats) 类抗性基因LRR区的保守模体,呈现XXLXXLXXL重复。首次将SON-PCR方法成功地运用到植物基因组学研究中，为植物基因克隆又提供一方法。

SON-PCR Cloning the Leucine-rich Repeats Region of Cereal Cyst Nematode Resistance Gene and Sequencing

According to the sequence of Rccn4 which has high similarity to the nucleotide binding site(NBS) coding region of cereal cyst nematode Heterodera avenae resistance gene, Cre3, three 3' nested primers were designed. Through single oligonucleotide nested PCR (SON-PCR), one band, Rccn-L of 1264 bp(GenBank accession No. DQ124933), was successfully amplified from E-10 which is the wheat-Aegilops variabilis translocation line containing the cereal cyst nematode resistance gene of A.variabilis. This band of interest was the 3' flanking sequence of Rccn4 and contained a common sequence of 55 bp with Rccn4. The coding region was 1026 bp, which contained an incomplete open reading frame and a terminator codon, without initiation codon and intron, encoding a peptide of 342 amino acid residues, and shared 86%nucleotide sequence identity with Cre3. This peptide had a conserved LRR(leucine-rich repeats) domain, containing the imperfect repeats,XXLXXLXXL, which contains 17% leucine residues and shares, respectively, 89% nucleotide sequence and 78% amino acid sequence identity with the LRR sequence of Cre3 locus. This research first successfully used SON-PCR in the research of plant genome, which indicated that SON-PCR is one more method for cloning plant gene.