改良高盐高pH法提取大豆DNA  

Extraction of Soybean DNA by Improved High Salt and High pH Method

作　　者：杨学珍 刘晓雪[1] 宋健 乔虎平 邹娟[4] 杨前玉 张帆[2] 王俊[1] YANG Xue-zhen;LIU Xiao-xue;SONG Jian;QIAO Hu-ping;ZOU Juan;YANG Qian-yu;ZHANG Fan;WANG Jun(College of Agronomy,Yangtze University,Jingzhou 434025,China;College of Life Sciences,Yangtze University,Jingzhou 434025,China;Biomarker Technologies,Beijing 103100,China;Hubei Academy of Agricultural Sciences,Wuhan 430064,China;Hubei Win All Hi Technology Seed Industry Co.,Ltd.,Jingzhou 434025,China)

机构地区：[1]长江大学农学院,湖北荆州434025 [2]长江大学生命科学学院,湖北荆州434025 [3]百迈客生物科技有限公司,北京101300 [4]湖北省农业科学院,湖北武汉430064 [5]湖北荃银高科种业有限公司,湖北荆州434025

出　　处：《大豆科学》2020年第4期549-554,共6页Soybean Science

基　　金：湿地生态与农业利用教育部工程研究中心开放基金(KF201808,KF201910)。

摘　　要：大豆除了含有大量的脂类和蛋白质外,还含有糖类和酚类物质,为解决难于从大豆中提取高质量DNA和传统提取方法耗时长的问题,本研究以中豆41与天隆一号的幼嫩叶片(V3)和成熟种子作为材料,对高盐低pH法进行改良,提高其裂解液pH值,同时结合冷冻裂解法缩短裂解时间,使用不同pH值醋酸钠纯化DNA,并与经典CTAB法进行比较,评价其提取DNA的效果及其应用范围。结果表明,高盐高pH法对成熟种子的DNA提取效率较高,且蛋白与RNA污染少。CTAB法提取的DNA虽然完整性较高,但是耗时较长,蛋白与RNA污染较多。另外,高盐高pH法提取的DNA PCR扩增条带清晰,与常规CTAB法提取DNA扩增效果一致,但其DNA质量不能满足高通量测序要求。综上,改良的高盐高pH法是一种快速有效的大豆DNA提取方法,可满足常规分子试验的要求。Soybean contains substantial amount of protein and lipids, and sugar and phenols as well, which made the nucleic acid(DNA/RNA) extraction rather difficult and traditional DNA extraction methods are tedious and time-consuming. In the present study, high salinity and low pH methods was improved by elevating the pH value of lysis solution using mature seeds and leaves of Zhongdou 41 and Tianlong 1, respectively. The extraction process was shorten by the application of repeated freeze pyrolysis and DNA was purified by acetate sodium of distinct pH. The improved high salinity and high pH method was then evaluated in terms of effect, efficiency, and application scope in comparison with CTAB method. Results showed that the DNA extraction efficiency of high salinity and high pH method from seeds was significantly higher than CTAB with less contamination of protein and RNA residuals, but with less DNA integrity. Whereas, the CTAB yielded DNA with relatively higher DNA integrity but with more contamination and slower extraction speed. Besides, during the PCR amplification, the DNA extracted by high salinity and high pH showed clearly bands, which was similar with that by CTAB, however, which can not meet the standard of high throughput sequencing. The improved DNA extraction method was suitable for rapid DNA extraction in soybean, which could meet the demand of daily-use molecular experiments.

关 键 词：大豆 DNA提取 高盐高pH CTAB 

分 类 号：S565.1[农业科学—作物学]