几个常用内参基因在淀粉酶产色链霉菌1628抗药性突变株中稳定性的评估

doi:10.16409/j.cnki.2095-039x.2016.04.014

中国生物防治学报 ›› 2016, Vol. 32 ›› Issue (4): 518-523.DOI: 10.16409/j.cnki.2095-039x.2016.04.014

几个常用内参基因在淀粉酶产色链霉菌1628抗药性突变株中稳定性的评估

汤谷, 姚佳忆, 申屠旭萍, 俞晓平

中国计量大学生命科学学院/浙江省生物计量及检验检疫技术重点实验室, 杭州 310018

收稿日期:2015-10-29 出版日期:2016-08-08 发布日期:2016-07-29
通讯作者: 俞晓平
作者简介:汤谷,男,硕士研究生,E-mail:305002848@qq.com
基金资助:
国家自然科学基金（31401793；31461143030）；浙江省自然科学基金（LY12C14012）；浙江省公益技术研究项目（2010C32063）

Evaluation of Several Endogenous Reference Genes in Streptomyces diastatochromogenes 1628 and Its Drug Resistance Mutants

TANG Gu, YAO Jiayi, SHENTU Xuping, YU Xiaoping

Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine/College of Life Science, China Jiliang University, Hangzhou 310018, China

Received:2015-10-29 Online:2016-08-08 Published:2016-07-29

摘要/Abstract

摘要： 淀粉酶产色链霉菌Streptomyces diastatochromogenes 1628是一株重要的生防菌株，其代谢产物对多种植物病原真菌具有较强的抑制效果。为了筛选在其不同抗药性高产突变株中均能稳定表达的内参基因，选择16S rRNA、sigB、hrdB、thyA、gyrB和rpoA共6个常见内参基因，分别探究它们在野生型菌株、链霉素抗性突变株、利福平抗性突变株和巴龙霉素抗性突变株中的表达情况，并用geNorm软件和NormFinder软件对结果进行分析。结果表明，使用不同软件分析得出的最佳内参基因略有差异。geNorm软件认为在上述4株菌株中，表达最稳定的内参基因分别是gyrB、gyrB、thyA、rpoA；而NormFinder软件认为rpoA基因在所有菌株的表达都是最稳定的。利用平均等级的算法平衡两个软件的分析差异，最终确定rpoA基因为表达最稳定的内参基因。通过检测toyG基因的表达水平，发现以rpoA作为内参基因能得到更合理的结果。

关键词: 淀粉酶产色链霉菌, 荧光定量PCR, 内参基因

Abstract: Metabolites of Streptomyces diastatochromogenes 1628 have strong inhibition on various plant pathogenic fungi. To screen out the most stably expressed endogenous reference genes in different drug resistance overproduction mutants of S. diastatochromogenes 1628, six candidate reference genes were selected for real-time quantitative PCR, and two kinds of software were adopted to determine the stability of candidate reference genes in different drug resistance strains. The results revealed that the optimum reference gene analyzed by different softwares was slightly different. Analysis of geNorm software showed the gyrB, gyrB, thyA, rpoA were the most stably expressed reference gene in wild-type, streptomycin-resistance, rifampin-resistance and paromomycin- resistance strain, respectively. However, rpoA was the most stable gene in tested strains analyzed by NormFinder. Comprehensively, the mean rank based on stability banking analyzed by these two kinds of software showed that rpoA was the most suitable reference gene in all tested strains. By detecting the expression level of toyG gene, it was found that rpoA as internal control gene is more reasonable.

Key words: Streptomyces diastatochromogenes, qPCR, reference gene

中图分类号:

S476

汤谷, 姚佳忆, 申屠旭萍, 俞晓平. 几个常用内参基因在淀粉酶产色链霉菌1628抗药性突变株中稳定性的评估[J]. 中国生物防治学报, 2016, 32(4): 518-523.

TANG Gu, YAO Jiayi, SHENTU Xuping, YU Xiaoping. Evaluation of Several Endogenous Reference Genes in Streptomyces diastatochromogenes 1628 and Its Drug Resistance Mutants[J]. journal1, 2016, 32(4): 518-523.

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几个常用内参基因在淀粉酶产色链霉菌1628抗药性突变株中稳定性的评估

Evaluation of Several Endogenous Reference Genes in Streptomyces diastatochromogenes 1628 and Its Drug Resistance Mutants

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