基于内转录间隔区和β-微管蛋白部分基因及相关序列扩增多态性分子标记技术推断红曲霉系统发育关系
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(1.国家食品安全风险评估中心 国家卫生健康委员会食品安全风险评估重点实验室,北京 100021;2.中国疾病预防控制中心传染病预防控制所,北京 102206)

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王美美 女 助理研究员 研究方向为食品微生物E-mail:wangmeimei@cfsa.net.cn通信作者:┣┣(中)通信作者┫┫李凤琴 女 研究员 研究方向为食品微生物E-mail:lifengqin@cfsa.net.cn

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Genetic diversity analysis of Monascus strains using internal transcribed space, partial β-tubulin gene and sequence-related amplification polymorphism markers
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(1.NHC Key Laboratory of Food Safety Risk Assessment, China National Center for Food Safety Risk Assessment, Beijing 100021, China;2.National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China)

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    摘要:

    目的 基于内转录间隔区(ITS)、β-微管蛋白(β-tubulin)部分基因和相关序列扩增多态性(SRAP)分子标记技术结合形态学鉴定方法,推断不同种红曲霉的系统发育关系,寻找快速、准确鉴定红曲霉的方法。方法 以红曲霉基因组为模板,聚合酶链式反应(PCR)扩增红曲霉ITS、β-tubulin部分基因,利用Mega 7.0软件中最大似然树法构建进化树,扩增SRAP分子标记技术的特征结合序列,利用R软件phangorn包中的FigTree软件进行建树,使用除权配对(UPGMA)法进行聚类分析,通过分析不同种红曲霉系统发育关系,找寻快速、准确的鉴定方法。结果 利用ITS和β-tubulin部分基因可将31株供试菌株分为两大类,而SRAP分子标记技术可将供试菌株分为四大类,结合表型分析和3种分子鉴定方法,参照15株红曲霉参考菌株将16株未鉴定到种的红曲霉分别鉴定为安卡红曲霉、橙色红曲霉和紫色红曲霉。结论 SRAP分子标记技术具有更多的分类依据,结合表型分析能够帮助更快、更精确的将红曲霉鉴定到种。

    Abstract:

    Objective Based on the sequence-related amplification polymorphism (SRAP) technology and the sequence similarity of internal transcribed space(ITS),β-tubulin and combined with morphological identification method, the phylogenetic relationships of different species of Monascus were inferred and a reliable method for rapid and accurate identification of Monascus was developed. Methods Using the Monascus genome as template, ITS and partial β-tubulin gene sequence were amplified and sequenced.Phylogenetic tree was constructed by using the maximum likelihood method with Mega 7.0 software. SRAP specific binding sequences were also amplified and the phylogenetic tree was built by using unweighted pair-group method with arithmetic means(UPGMA) algorithm with FigTree software in R phangorn package. A rapid and reliable identification method was built by analyzing the phylogenetic relationships of different species of Monascus. Results Combined with phenotypic analysis and two molecular identification method,16 strains of Monascus was identified as Monascus anka, Monascus aurantiacus and Monascus purpureus. Conclusion SRAP identification method had more detailed classification basis and could identify Monascus species quickly and accurately when combined with phenotypic analysis.

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王美美,张建中,李凤琴.基于内转录间隔区和β-微管蛋白部分基因及相关序列扩增多态性分子标记技术推断红曲霉系统发育关系[J].中国食品卫生杂志,2018,30(6):563-569.

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  • 收稿日期:2018-11-14
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  • 在线发布日期: 2019-01-23
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