易变山羊草抗禾谷孢囊线虫基因中核苷酸结合位点区(NBS)的克隆及序列分析

Cloning and Sequencing of the Coding Region of Nucleotide-binding Site in Cereal Cyst Nematode Resistance Gene from Ae.variabilis

大部分已克隆的植物抗病基因都包含有核苷酸结合位点区 (NBS)和富含亮氨酸的重复序列区 (LRR) .利用来自节节麦的抗禾谷孢囊线虫基因Cre3位点NBS区保守序列设计特异引物 ,从含有来自易变山羊草的抗禾谷孢囊线虫基因的小麦品系E 10的基因组中PCR扩增得到一条约 5 30bp的单一条带 .将扩增条带克隆和序列分析发现 ,该克隆 (Rccn4 )的编码区长 5 2 8bp ,含一个不完整的开放读码框 ,没有起始密码子、终止密码子和内含子结构 ,它编码一个 176个氨基酸残基的蛋白质 ,分子量为 2 0 4kD .Rccn4含有NBS LRR类抗病基因NBS区共有的保守模体I(V)LDD、T(T S)R、G(L S)PLA(A I L)、RCF(A L)Y ,并且与Cre3基因的NBS编码区核苷酸和氨基酸同源性分别为99 4 %和 98% .

Most of plant disease resistance (R) genes cloned so far belong to NBS LRR group which contains nucleotide binding sites (NBS) and a leucine rich repeat (LRR). Specific primers were derived from the conserved motif of NBS sequence at the Cre3 locus, which conferred resistance to cereal cyst nematode (CNN) in the wild wheat ( Triticum tauschii ),and the specific primers were used in isolating a cereal cyst nematode resistance gene through a polymerase chain reaction (PCR) cloning approach. Only one band of approximately 530 bp in size was amplified from wheat E 10 conferring the resistance to cereal cyst nematode from Ae.variabilis . This target fragment was cloned and sequenced,and coding region of this clone(Rccn4)was 528 bp in size,which contained an incomplete open reading frame without initiation codon ,terminator codon and exon encoding a peptide of 176 amino acid residues .The molecular weight of the protein from the amino acid was 20 4 kD. The amino acid sequence of Rccn4 contained conserved motifs :I(V)LDD ,T(T/S)R , G(L/S)PLA(A/I/L)and RCF(A/L)Y, present in known R gene containing NBS from other plants.Rccn4 shares 99 4 % nucleotide sequence and 98 % amino acid sequence identity with the NBS sequence at the Cre3 locus. It is a novel gene containing NBS resistance to cereal cyst nematode.