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益生菌FGM葡萄糖激酶基因（glcK）在黄芪发酵中的表达

ISSN号：1004-1389
期刊名称：西北农业学报
时间：2013
页码：1-5
分类：S853.2[农业科学—临床兽医学;农业科学—兽医学;农业科学—畜牧兽医]
作者机构：[1]中国农业科学院兰州畜牧与兽药研究所,甘肃省中兽药工程技术研究中心,甘肃省新兽药工程重点实验室,农业部兽用药物创制重点实验室,兰州730050
相关基金：国家自然科学基金（31072162）; 国家行业农业科研专项（201303040）; 公益性研究所基本科研费（1610322011007）
相关项目：乳杆菌FGM9体外转化黄芪多糖的机理研究

关键词：黄芪, 非解乳糖链球菌, 发酵, 葡萄糖激酶, 基因克隆, 实时荧光定量PCR, Astragalus membranaceus, Streptococcus alactolyticus, Fermentation, Glucokinase, Gene cloning, Real-time PCR

中文摘要：

旨在分析益生菌FGM在黄芪发酵过程中的生长动态,为阐述其作用机理奠定基础。采用平板计数法对益生菌FGM在黄芪发酵中的生长动态进行检测,同源克隆法克隆糖酵解途径中的限速酶葡萄糖激酶glcK基因序列,并运用SYBR GreenⅠ实时荧光定量PCR技术分析其在黄芪发酵不同阶段的表达水平。结果表明：成功克隆得到glcK基因一段382bp的片段（GenBank登录号JX976291）,其序列一致性为83%～85%。实时荧光定量PCR结果显示,在FGM发酵6h内,glcK基因表达上调且呈逐渐上升趋势,至对数期后期6h表达水平达到最高（4.63倍）;稳定期初期8h时表达水平显著下降（P〈0.05）,10h至发酵结束基因表达下调。可见：在黄芪发酵6h内FGM处于对数生长期,葡萄糖经糖酵解途径代谢产生ATP供细菌生长代谢利用,10h后开始进入稳定期并进行次级代谢。

英文摘要：

This study was aimed at analyzing the growth of probiotic FGM in Astragalus membranaceus fermentation to lay a foundation for understanding the mechanism.FGM growth dynamics in A.membranaceus fermentation were investigated with plate count method.The nucleotide sequence of glucokinase catalyzing the rate-limiting step of the glycolysis pathway was cloned and the variations of glcK gene expression during fermentation process was further analyzed by SYBR Green real-time PCR.As a result,a segment of 382 bp（GenBank accession No.JX976291）,displaying high sequence identity from 83% to 85%,was successfully obtained for the first time.Real-time PCR results showed that glcK gene expression was up-regulated and increased gradually within 6 h during A.fermentation.The expression level was peaked at 6 h and declined significantly at 8 h,and thereafter glcK gene expression was down-regulated from 10 h to the end.Those results demonstrated that FGM was in exponential growth phase within 6 h,when glucose was metabolized for bacteria growth by the glycolysis pathway metabolism.After 10 h of fermentation,FGM was in stable phase for secondary metabolism.

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