赵芹等

摘 要 以黑皮冬瓜“B98K”种质为试材，根据已克隆的植物NBS类抗病基因保守结构域设计简并引物，PCR扩增冬瓜基因组DNA，获得对应区段的DNA片段，回收克隆与测序后，得到19条抗病同源序列（命名为DNB1至DNB19）。利用DNAStar软件进行序列分析及同源性比对发现，这些抗病同源序列长度在249～252 nt之间，其核苷酸序列同源性在48.4%～98.8%之间，氨基酸序列同源性为23.2%～100.0%，推导氨基酸序列具有P-loop（GGVGKTT）、Kinase-2a（VLDDVW）典型的NBS保守结构域。同源进化分析将其全部归为nonTIR-NBS-LRR 类，与推导氨基酸序列多重比较结果一致；该序列与其他作物已克隆抗病基因的氨基酸序列同源性在24.7%～99.0%之间。该组序列已登录Genbank，登录号为KF776401～KF776419。

关键词 冬瓜；NBS；抗病同源序列；同源克隆；序列分析

中图分类号 S642 文献标识码 A

Abstract According to the conserved domains of NBS type disease-resistant genes in most known plants， two degenerate primers were designed and synthesized. Nineteen RGAs named from DNB1 to DNA19 were obtained by PCR amplification of genome DNA from black wax gourd“B98K”variety. Sequences analysis and alignment results indicated that full-length of these RGAs varied from 249nt to 252nt， the deduced amino acids sequences contained typical conserved domains of NBS family， such as P-loop（GGVGKTT）and Kinase-2a（VLDDVW）. The GenBank accession numbers were obtained（from KF776401 to KF776419）. These RGAs showed great homologous differences with the similarity from 48.4% to 98.8% and with genetic divergence from 1.2 to 88.9 in nucleotide sequence level， while the amino acid sequence homology varied from 23.2% to 100.0%. These RGAs were all divided into nonTIR-NBS-LRR group， which was consistent with the multiple-alignment of deduced amino acid sequences. The similarities varied from 24.7% to 99.0% compared with NBS RGAs of other known species.

Key words Ax gourd； NBS； Resistance gene analogs； Homologous clone； Sequence analysis

doi 10.3969/j.issn.1000-2561.2014.10.031

冬瓜（Benincasa hispida Cogn.）属葫芦科冬瓜属1年生蔓性植物，原产中国与印度，现广泛分布于亚洲的热带、亚热带和温带地区。在中国已有2 000多年的栽培历史，南北地区均有种植，年播种面积达25万hm2[1]。冬瓜易栽培，产量高，耐贮运，保健药用价值高[2-3]，是夏秋季节的重要蔬菜之一，也是华南地区北运和出口创汇的重要蔬菜品种，对于调节蔬菜淡季、保证蔬菜周年供应起着非常重要的作用[4]。但冬瓜的各种病害成为制约冬瓜生产的重要因素，其中枯萎病、疫病以及病毒病为冬瓜3大主要病害，南方地区高温多雨，发病更加严重和频繁，损失异常严重[5]。本课题组通过多年种质资源引进、收集和创新，拥有近400份不同抗性冬瓜材料，并通过病原菌复合接种获得了2份冬瓜多抗优异材料[5]，利用本课题组的资源优势，开展冬瓜抗病基因挖掘，分离鉴定与冬瓜抗病相关的基因序列，将为冬瓜抗病品种选育提供理论基础和技术支持。……