摘要
食用菌细胞壁的复杂性增加了提取基因组的难度,为找到满足真姬菇全基因组序列分析的高质量基因组提取方法,分别使用氯仿-Tris法与CTAB-SDS法等12种方法提取真姬菇菌丝体基因组,并进行样品前处理的优化。经琼脂糖凝胶电泳、紫外分光光度计测定浓度和纯度、ITS序列的PCR扩增检测,结果表明:12种方法提取真姬菇菌丝体基因组时,氯仿-Tris法(样品烘干处理)与CTAB-SDS法(样本烘干处理)能得到较为完整的DNA。另外,通过在氯仿-Tris法(样品烘干处理)与CTAB-SDS法(样本烘干处理)的基础上增加柠檬酸钠-乙醇洗涤步骤,得到优化CTAB-SDS法。PCR检测结果显示,3种方法所得基因组DNA均能有效进行相应的酶反应。综合考虑基因组的浓度、纯度、完整性和有无降解等因素,优化CTAB-SDS法提取得到的基因组DNA(浓度为408.7 ng/μL,OD_(260)/OD_(280)为1.81±0.02)最适于真姬菇全基因组的序列分析。
The complexity of fungal cell wall increase the difficulty of extracting genome. In order to find a suitable DNA extracting method for the whole genome sequencing (WGS) of Hypsizygus marmoreus (Peck) H.E.Bigelow, twelve methods including the chloroform-Tris, the CTAB-SDS, and so on were testified. In the meantime the sample pretreatment were also optimized. The DNA concentration and purity were determined by agarose gel electrophoresis and UV spectrophotometer. PCR amplification of the ITS sequences were also verified. The results showed that the chloroform-Tris (sample drying) and the CTAB-SDS (sample drying) could obtain intact genome of H. marmoreus. Moreover, modified CTAB-SDS method was then obtained from the chloroform-Tris (sample drying) and the CTAB-SDS (sample drying) by adding Na-citrate-ethanol washing. The results from the PCR showed that, the genome from the three methods can all react with enzymes. In general, considering the concentration, the purity and the integrity, the DNA from modified CTAB-SDS (concentration: 408.7 ng/μL, OD260/OD280:1.81±0.02) was the optimized method for WGS of H. marmoreus.
作者
孙溪
班立桐
王玉
肖萍
范志华
王旭峰
杨华
黄亮
SUN Xi;BAN Litong;WANG Yu;XIAO Ping;FAN Zhihua;WANG Xufeng;YANG Hua;HUANG Liang(College of Biological Engineering,Tianjin Agricultural University,Tianjin 300384;College of Agronomy and Resource Environment,Tianjin Agricultural University,Tianjin 300384;Tianjin Lvshengpengyuan Agricultural Science and Technology Development Co.LTD.,Tianjin 300400)
出处
《食品科技》
CAS
北大核心
2018年第12期31-36,共6页
Food Science and Technology
基金
天津市特支计划-青年拔尖人才项目(TJTZJH-QNBJRC-2-12)
天津市蔬菜产业技术体系创新团队项目(ITTVRS2017015)
国家自然科学基金项目(31701569)
天津市北辰区农业科技发展项目(2017-NYKJ-03)
作者简介
孙溪(1981-),女,天津人,博士研究生,研究方向为工业育种;通信作者:黄亮
