Acta Agrestia Sinica

Acta Agrestia Sinica ›› 2016, Vol. 24 ›› Issue (5): 1073-1079.DOI: 10.11733/j.issn.1007-0435.2016.05.022

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Polymorphism Analysis on Polygalacturonase-inhibiting Protein 2 (MsPGIP2) Gene in Alfalfa

GUI Zhi1, PI Yong-shuo1, YUAN Qing-hua2, QU Ze-peng1, GAO Jian-ming1,2   

  1. 1. College of Agronomy, Resources and Environmental Science, Tianjin Agricultural University, Tianjin 300384, China;
    2. Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China
  • Received:2015-11-11 Revised:2016-03-15 Online:2016-10-15 Published:2017-01-13

苜蓿多聚半乳糖醛酸酶抑制蛋白2(MsPGIP2)基因多态性分析

桂枝1, 皮永硕1, 袁庆华2, 曲泽鹏1, 高建明1,2   

  1. 1. 天津农学院 农学与资源环境学院, 天津 300384;
    2. 中国农业科学研究院 北京畜牧兽医研究所, 北京 100193
  • 通讯作者: 高建明
  • 作者简介:桂枝(1973-),女,山西太原人,硕士,副教授,主要从事饲用作物遗传育种研究,E-mail:guizhi73@163.com
  • 基金资助:

    国家自然科学基金（青年）项目（No.31201840）；天津市高等学校创新团队培养计划（No.TD12-5017）资助

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Abstract:

This study aimed to isolate the full-length genomic sequence of polygalacturonase-inhibiting protein 2 (MsPGIP2) gene and analyze its sequence variations. Genome walking method based on the thermal asymmetric PCR using homologous primers was used to isolate the DNA sequence of MsPGIP2 gene, and the polymorphisms among the three alfalfa cultivars were detected by clone sequencing. The results showed that the full-length genomic sequence of the gene had a size of 1 126 bp, and could be divided into three regions including signal peptide, intron and leucine-rich repeat (LRR) region. A total of 46 variation sites were found in the MsPGIP2 sequence (frequency >0.02), but no variation site was detected in signal peptide. Intron contained five single nucleotide polymorphism (SNP) sites and two InDel sites while LRR region contained 39 SNP sites, of which, 61.5% belonged to non-synonymous mutations. Furthermore, 15 alleles, namely haplotypes, were detected among the studied cultivars and they were grouped three clusters by phylogenetic analysis. In conclusion, MsPGIP2 gene has great sequence variations due to frequent hybridization among its alleles, and therefore evoluted quickly to adapt to the quick evolution of the polygalacturonase from pathogens.

Key words: Medicago sativa, Gene of polygalacturonase-inhibiting protein, Single nucleotide polymorphism

摘要：

本研究旨在分离苜蓿多聚半乳糖醛酸酶抑制蛋白2（MsPGIP2）基因的基因组序列全长，分析序列变异。首先采用引物热不对称PCR的基因组DNA步移法分离其基因组序列全长，然后通过克隆测序分析了MsPGIP2基因在3个苜蓿品种（‘润布勒’、‘苏普斯坦’与‘公农1号’）中的多态性。结果显示，MsPGIP2基因的基因组DNA序列全长1 126 bp，可分为信号肽、内含子和LRR区3部分。MsPGIP2基因的基因组序列中共有46个变异位点（频率>0.02）。在信号肽区，没有变异位点；在内含子区，有5个SNP位点和2个InDel变异位点；而在LRR区，共发现了39个SNP位点，其中非同义突变占61.5%。在3个苜蓿品种中共发现了15个等位基因（即单倍型），通过对LRR区核苷酸的系统发育分析将他们分成3类。结论认为，MsPGIP2基因具有较大的变异，各等位基因间发生了频繁的重组交换，是一个为适应病原微生物PG的进化而快速进化的基因。

关键词: 紫花苜蓿, PGIP基因, 单核苷酸多态性

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