食品科学 ›› 2013, Vol. 34 ›› Issue (7): 206-211.doi: 10.7506/spkx1002-6630-201307043

• 生物工程 • 上一篇    下一篇

牦牛奶酪中乳酸菌的随机扩增多态性研究

杨吉霞1,陈芝兰2,杨海燕3,黄艾祥4,陈宗道1   

  1. 1.西南大学食品科学学院,重庆 400715;2.西藏农牧学院生物技术中心,西藏 林芝 860000; 3.新疆农业大学食品科学与药学学院,新疆 乌鲁木齐 830052;4.云南农业大学食品科学与技术学院,云南 昆明 650201
  • 收稿日期:2012-07-15 修回日期:2013-03-12 出版日期:2013-04-15 发布日期:2013-03-20
  • 通讯作者: 杨吉霞 E-mail:yangjx@swu.edu.cn
  • 基金资助:
    2009年西南大学中央高校基本科研业务费专项(XDJK2009C039);国家自然科学基金地方项目(31060020)

Randomly Amplified Polymorphic DNA (RAPD) Analysis of Lactic Acid Bacteria from Yak Milk Cheese

YANG Ji-xia1,CHEN Zhi-lan2,YANG Hai-yan3,HUANG Ai-xiang4,CHEN Zong-dao1   

  1. 1. College of Food Science, Southwest University, Chongqing 400715, China; 2. Center of Biology, Tibet Agricultural and Animal Husbandry College, Linzhi 860000, China; 3. College of Food Science and Pharmacy, Xinjiang Agricultural University, ürümqi 830052, China; 4. College of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, China
  • Received:2012-07-15 Revised:2013-03-12 Online:2013-04-15 Published:2013-03-20
  • Contact: Ji-Xia YANG E-mail:yangjx@swu.edu.cn

摘要: 目的:探讨随机扩增多态性(RAPD)技术在牦牛奶酪中乳酸菌基因型研究方面的应用价值。方法:采用M13引物,对退火温度、Mg2+浓度、引物用量、Taq酶用量、模板量进行单因素梯度试验,建立最佳反应条件,然后对分离自牦牛奶酪的39株乳酸菌进行扩增,用NTsys 2.10e软件分析扩增图谱,计算遗传相似系数,进行聚类分析,并与16S rRNA测序得到的菌种鉴定结果进行对比。结果:39株乳酸菌相互间的遗传相似系数在0.47~0.98之间,当相似系数为0.78时,菌株被分成10个组,除X23、Y36和Y37之外,其余菌株均按照不同的菌种聚类,与16S rRNA测序结果基本一致,聚类有较强的地域相关性。结论:RAPD技术可用于牦牛奶酪中乳酸菌的基因型研究,可用于菌种鉴定和遗传亲缘关系分析。

关键词: 牦牛奶酪, 乳酸菌, 随机扩增多态性, 聚类分析, 基因型鉴定, 遗传相似系数

Abstract: Objective: To explore the application value of randomly amplified polymorphic DNA (RAPD)-PCR for the genotype evaluation of lactic acid bacteria isolated from yak milk cheeses. Methods: In order to establish a reliable RAPD protocol with primer M13, factors such as annealing temperature, Mg2+ concentration, primer amount, Taq polymerase and DNA template for affecting the quality and reproducibility of RAPD profiles were investigated. The optimal PCR procedure and reaction reagent concentration were employed for RAPD amplification of 39 isolates from yak milk cheese on the basis of previous identification of 16S rRNA sequence analysis. The electrophoretic profiles were analyzed using NTsys2.10e software, genetic similarity coefficients were calculated and a cluster dendrogram was plotted. Results: Genetic similarity coefficients of 39 strains ranged from 0.47 to 0.98. The cluster dendrogram showed that 39 isolates were classified into 10 groups using the similarity coefficient of 0.78 as a cut-off point. The discrimination of groups was consistent with the assignment of the species based on 16S rRNA sequence analysis, except for strains X23, Y36 and Y37. Conclusion: RAPD technique is useful in taxonomy of lactic acid bacteria isolated from yak milk cheese. The accuracy of species identification can be improved by the combination of RAPD with other kinds of genotypic methods.

Key words: yak milk cheese;lactic acid bacteria;randomly amplified polymorphic DNA (RAPD);cluster analysis, genotype;genetic similarity coefficient

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