Cloning of resistance gene analogs from Lagenaria siceraria based on conserved domains of NBS -LRR type R gene
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摘要:目的
分离鉴定瓠瓜Lagenaria siceraria抗病种质的抗病基因同源序列(Resistance gene analogs, RGAs), 为瓠瓜功能性抗病基因的克隆及分子标记辅助育种奠定基础.
方法根据已知核苷酸结合位点和富亮氨酸重复(Nucleotide binding site and leucine rich repeat, NBS-LRR)类抗病基因保守区设计简并引物, 从“大籽瓠”抗性材料基因组DNA中分离抗病基因同源序列, 并利用生物信息学软件进行长度变异、保守结构域、同源比对与系统进化分析.
结果和结论基于同源克隆策略, 获得23条瓠瓜NBS抗病同源序列, 命名为HNB1 ~ HNB23, GenBank登录号为KJ908192 ~ KJ908214.序列分析及同源比对结果表明, 这些RGAs长度变异在242 ~ 261 nt之间, 18条序列具有连续开放阅读框(Open reading frame, ORF), 推导氨基酸序列具有P-loop、Kinase-2a典型NBS类R基因保守结构域；核苷酸序列相似性为41.5% ~ 98.8%, 氨基酸序列相似性为21.5% ~ 100.0%；利用NCBI Blast同源搜索发现, 与其他植物尤其是冬瓜、黄瓜、葫芦及丝瓜的已知R基因具有40% ~ 100%相似性；氨基酸序列聚类分析将其分为5个组；同源进化分析表明, 23条瓠瓜RGAs均为non-TIR-NBS-LRR类R基因, 与推导氨基酸序列多重比较结果一致.
Abstract:ObjectiveIsolation and identification of resistance gene analogs (RGAs) of Lagenaria siceraria would lay the foundation for a further cloning of disease resistance genes and marker-assisted selection (MAS) of resistance breeding.
MethodAccording to the conserved domains of nucleotide binding site and leucine rich repeat (NBS-LRR) type of disease-resistance genes in most known plants, degenerate primers were designed and synthesized to isolate resistance gene analogs from genomic DNA of bottle gourd resistant variety " Dazihu", with length variation, conservative domain, homology alignment and phylogenetics analyzed by various bioinformatics softwares.
Result and conclusionTwenty three RGAs were obtained and named as HNB1 - HNB23, and the GenBank accession numbers were KJ908192 - KJ908214.Sequences analyses and alignment results indicated that the full-length of RGAs varied from 242 nt to 261 nt, and the deduced amino acids sequences contained typical conserved domains of NBS R genes, such as P-loop and Kinase-2a.Eighteen sequences had continuous open reading frames (ORFs).These RGAs showed a great homologous differences with the similarity ranging from 41. 5% to 98.8%, and the amino acid sequence similarity varied from 21.5% to 100.0%.At the nucleotide level, the sequence identity of 23 RGAs ranged from 40% to 100% with the cloned NBS R genes from other plants, especially cucumber, wax gourd, luffa and calabash.The result of clustering analyses showed that all RGAs were divided into 5 groups.These RGAs were ranked into non-TIR-NBS-LRR type by homology and evolution analyses, which was consistent with the classification result based on multiple alignment of deduced amino acid sequences.
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Keywords:
- Lagenaria siceraria /
- NBS-LRR type /
- resistance gene analogs /
- degenerate primer /
- sequence analysis
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图 1 瓠瓜NBS类RGAs PCR产物电泳分析
Figure 1. Electrophoreses analyses of PCR products of NBS RGAs from bottle gourd
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图 2 瓠瓜NBS类RGAs编码氨基酸序列的聚类分析
Figure 2. Clustering analyses of amino acid sequences encodcd by NBS type RGAs in bottle gourd
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图 3 瓠瓜NBS类RGAs编码氨基酸序列比对
“-”用于优化联配的缺口；下划线标注为NBS R基因保守功能域“ P-loop”与“ Kinase-2”.
Figure 3. Amino acid sequence alignments of NBS type resistance RGAs in bottle gourd
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