浓香型白酒酒醅中总RNA提取方法评价

doi:10.7506/spkx1002-6630-20191110-106

食品科学 ›› 2021, Vol. 42 ›› Issue (2): 74-82.doi: 10.7506/spkx1002-6630-20191110-106

浓香型白酒酒醅中总RNA提取方法评价

胡晓龙，王康丽，宋丽丽，侯建光，曹振华，吴丽丽，牛广杰，马歌丽，赵书民，赵东

（1.郑州轻工业大学食品与生物工程学院，河南郑州 450000；2.河南仰韶酒业有限公司博士后研发基地，河南渑池 472000；3.河南宋河酒业股份有限公司，河南鹿邑 477200；4.河南省寿酒有限公司，河南新乡 453000；5.河南省酒业协会，河南郑州 450000；6.五粮液集团有限公司，四川宜宾 644000）

出版日期:2021-01-18 发布日期:2021-01-27
基金资助:
国家自然科学基金青年科学基金项目（31801535）；河南省重大科技专项（181100211400）；中国轻工业浓香型白酒固态发酵重点实验室开放基金项目（2017JJ012；2018JJ017）；郑州轻工业学院博士科研启动基金项目（2016BSJJ018）

Comparative Assessment of Modified Sodium Dodecyl Sulfate-Phenol Method and Other Methods for Total RNA Extraction from Fermented Grains of Chinese Strong-Flavor Baijiu

HU Xiaolong, WANG Kangli, SONG Lili, HOU Jianguang, CAO Zhenhua, WU Lili, NIU Guangjie, MA Geli, ZHAO Shumin, ZHAO Dong

(1. School of Food and Bio-engineering, Zhengzhou University of Light Industry, Zhengzhou 450000, China; 2. Postdoctoral Research & Development Base, Yangshao Distillery Co. Ltd., Mianchi 472000, China; 3. Henan Songhe Liquor Co. Ltd., Luyi 477200, China; 4. Henan Shoujiu Co. Ltd., Xinxiang 453000, China; 5. Henan Province Alcoholic Industry Association, Zhengzhou 450000, China; 6. Wuliangye Group Co. Ltd., Yibin 644000, China)

Online:2021-01-18 Published:2021-01-27

摘要/Abstract

摘要： 为探寻我国特有的复杂微生态环境——浓香型酒醅样品总RNA提取方法，本实验建立了一种改良的十二烷基硫酸钠（sodium dodecyl sulfate，SDS）-苯酚法，并从提取成本及耗时、RNA质量浓度和纯度、电泳结果、反转录效果及高通量测序分析5 个方面，比较其与试剂盒法、Trizol法、十六烷基三甲基溴化铵（hexadecyl trimethyl ammonium bromide，CTAB）法和月桂酸钠法提取酒醅样品总RNA的效果。结果表明，SDS-苯酚法和CTAB法成本较低，试剂盒法成本最高但耗时最短；试剂盒法提取的总RNA质量浓度最高，分别约为SDS-苯酚法、Trizol法、CTAB法和月桂酸钠法的1.84、2.33、19.14 倍和3.09 倍；SDS-苯酚法和试剂盒法提取样品总RNA的OD260 nm/OD280 nm和OD260 nm/OD230 nm均分别大于1.8和2.0；SDS-苯酚法电泳条带完整性优于其他方法，CTAB法无法检测到电泳条带。SDS-苯酚法、试剂盒法和Trizol法所提取的RNA能有效反转录为cDNA并将其用于高通量测序分析，3 种方法所得优势微生物种属（Lactobacillus和Clostridium）及不同发酵节点酒醅之间的微生物群丰度变化情况基本一致，其中SDS-苯酚法获得的微生物群落多样性最高，且能较好地反映不同酒醅间微生物类群的差异；Trizol法和试剂盒法均能偏好性地多获得1 个属，但其含量均很低（0.01%）。本研究系统地比较了5 种常见的总RNA提取方法在浓香型酒醅RNA提取的应用效果，其中本研究建立的改良SDS-苯酚法具有一定的综合优势，为今后以RNA为基础的酒醅分子生物学研究提供理论依据，同时对其他富含糖、腐殖质及酚类等杂质的环境样品RNA提取具有较好的借鉴意义。

关键词: 浓香型白酒酒醅；宏转录组；总RNA提取；提取方法评价；微生物群落

Abstract: A modified sodium dodecyl sulfate (SDS)-phenol method was proposed for total RNA extraction from fermented grains of Chinese strong-flavor Baijiu, a complex microecological environment unique to China. This method was compared with other common methods including commercial kit, Trizol method, hexadecyl trimethyl ammonium bromide (CTAB) method and sodium laurate method in regard to cost, extraction time, RNA concentration and purity, electrophoresis results, reverse transcription efficiency and high-throughput sequencing analysis. The results showed that the SDS-phenol and CTAB methods were inexpensive, while the kit method was the most time saving but most expensive. The total RNA concentration extracted by the kit method was the highest, which was 1.84, 2.33, 19.14 and 3.09 times as high as that by the modified SDS-phenol, Trizol, CTAB and sodium laurate methods, respectively. The OD260 nm/OD280 nm and OD260 nm/OD230 nm ratio of total RNA extracted by the SDS-phenol and kit methods were greater than 1.8 and 2.0, respectively. The electrophoresis showed more intact bands for total RNA extracted by the SDS-phenol method as compared to that extracted by the other methods, and but no bands for total RNA extracted the by the CTAB method. RNA extracted by the modified SDS-phenol, kit and Trizol methods could be efficiently reversely transcribed into cDNA and used for high-throughput sequencing analysis. The sequencing results showed that the dominant microbial genera (Lactobacillus and Clostridium) obtained by the three RNA extraction methods and the change of microbial population abundance in fermented grains at different fermentation times were basically the same. Among the three methods, the SDS-phenol method gave the highest diversity of microbial communities and could be useful to determine the differences in microbial groups among fermented grains at different fermentation times. Both the Trizol and kit methods could preferentially detect one more genus, but at very low levels (0.01%). In general, the modified SDS-phenol method was advantageous over the other RNA extraction methods. This study provides a theoretical basis for molecular biological research on fermented grains of Chinese Baijiu based on RNA in the future, and it is of reference significance for RNA extraction from environmental samples rich in sugar, humus and phenols.

Key words: fermented grains of Chinese strong-flavor Baijiu; metatranscriptome; total RNA extraction; evaluation of extraction methods; microbial community

中图分类号:

TS261

胡晓龙，王康丽，宋丽丽，侯建光，曹振华，吴丽丽，牛广杰，马歌丽，赵书民，赵东. 浓香型白酒酒醅中总RNA提取方法评价[J]. 食品科学, 2021, 42(2): 74-82.

HU Xiaolong, WANG Kangli, SONG Lili, HOU Jianguang, CAO Zhenhua, WU Lili, NIU Guangjie, MA Geli, ZHAO Shumin, ZHAO Dong. Comparative Assessment of Modified Sodium Dodecyl Sulfate-Phenol Method and Other Methods for Total RNA Extraction from Fermented Grains of Chinese Strong-Flavor Baijiu[J]. FOOD SCIENCE, 2021, 42(2): 74-82.

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浓香型白酒酒醅中总RNA提取方法评价

Comparative Assessment of Modified Sodium Dodecyl Sulfate-Phenol Method and Other Methods for Total RNA Extraction from Fermented Grains of Chinese Strong-Flavor Baijiu

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