随机扩增多态性法分析西藏牦牛奶酪中乳酸菌的遗传多样性

doi:10.7506/spkx1002-6630-201423042

摘要/Abstract

摘要：

目的：利用随机扩增多态性（randomly amplified polymorphic DNA，RAPD）法对西藏地区牦牛奶酪中27 株乳酸菌基因型进行同源性分析。方法：利用5 个随机引物对Mg2+浓度、dNTP用量、退火温度、模版用量/引物用量（ng/pmol）4 个条件做单因素梯度试验，建立最佳反应条件，筛选最佳引物，然后对27 株乳酸菌和4 株乳酸菌标准菌株进行随机扩增，用NTsys 2.10e软件对扩增条带进行聚类和遗传相似性系数分析，分析结果与16S rRNA测序得到的菌种鉴定结果进行对比。结果：31 株菌遗传相似系数在0.72～1.00之间，当相似性系数在0.82时，菌株被分成了8 组，菌株按照不同种属聚类，聚类结果同16S rRNA测序结果基本一致，同时成功将Lactobacillus casei和Lactobacillus paracasei两个亚种区分开。结论：RAPD技术可以较好地应用于西藏地区牦牛奶酪中乳酸菌亲缘性关系分析。

关键词: 西藏, 牦牛奶酪, 乳酸菌, 随机扩增多态性, 聚类分析

Abstract:

Objective: This study analyzed the homology of 27 strains of lactic acid bacterial genotype from Tibetan yak
cheese by randomly amplified polymorphic DNA (RAPD) method. Methods: To establish a reliable RAPD protocol with
5 primers, the optimal primers and PCR conditions for the quality and reproducibility of RAPD profiles were investigated,
including Mg2+ concentration, dNTP amount, annealing temperature, and DNA template/primer ratio. The optimal PCR
procedure and reaction reagent concentration were employed for RAPD amplification of 27 isolates from yak milk cheeses
and 4 standard strains of lactic acid bacteria. Electrophoretic profiles were analyzed by NTsys 2.10e software, genetic
similarity coefficients were calculated and a cluster dendrogram was plotted. Results: Genetic similarity coefficients of 31
strains ranged from 0.72 to 1.00. The cluster dendrogram showed that 31 isolates were classified into 8 groups using the
similarity coefficient of 0.82 as a cut-off point. The group discrimination corresponded well with the species assignment by
16S rRNA sequence analysis, and Lactobacillus casei and Lactobacillus paracasei were separated successfully. Conclusion:
The RAPD technique was useful for relationship analysis of lactic acid bacteria isolated from Tibetan yak milk cheeses.

Key words: Tibet, yak milk cheese, lactic acid bacteria, randomly amplified polymorphic DNA (RAPD), cluster analysis

中图分类号:

TS252.54

蒋厚阳1，赵国华1,2，杨吉霞1,2. 随机扩增多态性法分析西藏牦牛奶酪中乳酸菌的遗传多样性[J]. 食品科学, doi: 10.7506/spkx1002-6630-201423042.

JIANG Hou-yang1, ZHAO Guo-hua1,2, YANG Ji-xia1,2. Randomly Amplified Polymorphic DNA Analysis of Lactic Acid Bacteria from Yak Milk from Tibet Area[J]. FOOD SCIENCE, doi: 10.7506/spkx1002-6630-201423042.

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随机扩增多态性法分析西藏牦牛奶酪中乳酸菌的遗传多样性

Randomly Amplified Polymorphic DNA Analysis of Lactic Acid Bacteria from Yak Milk from Tibet Area

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