摘要
甘蔗是世界上重要的糖料作物和能源作物。目前,甘蔗分子生物学研究已成为甘蔗研究的热点之一。基因组DNA的提取是进行甘蔗分子生物学研究的基础。本研究设计含一系列SDS浓度的提取液,同时设加液氮和不加液氮研磨的对比试验,提取甘蔗不同部位叶片的基因组DNA并进行产量和纯度检测以及分子生物学分析。结果表明,所有提取液提取的甘蔗基因组DNA纯度均很高,A260/A280在1.8-2.0之间,A260/A230大于2,但提取液I(0.75%SDS)提取的甘蔗基因组DNA产量较低;加液氮与否对甘蔗基因组DNA的提取产量和纯度没有影响;以提取的甘蔗基因组DNA为模板,分别用一对扩增SPS(蔗糖磷酸合成酶)基因部分片段的引物和一对ISSR引物进行PCR扩增,所有DNA均能扩增出预期的条带;用不同的限制性内切酶对所提取的甘蔗基因组DNA进行酶切,所有DNA样品均能完全酶切。本研究得出最佳甘蔗基因组DNA提取方法如下:磨碎甘蔗叶片后,加DNA提取液(SDS:1.5%;Tris:100 mM;EDTA:20 mM;NaCl:500 mM)于65℃裂解30 min,经酚∶氯仿和氯仿各抽提一次,可获得高产量高质量的甘蔗基因组DNA,能满足后续分子生物学研究的要求。
Sugarcane is an important kind of sugar crop and energy crop in the world. Nowadays,research in molecular biology of sugarcane had become one of the focuses. And isolation of genomic DNA is the basic operation for molecular biology study on sugarcane. In this study,genomic DNA was isolated from different position leaves of sugarcane with solutions containing a series of concentrations of SDS. Meanwhile, grinding with or without liquid nitrogen were compared during isolation procedure. The quality and yield of all genomic DNA was detected,and analysis on molecular level was conducted. Detection of OD value showed that the quality of all DNA were high ,because the values of A260/A280 of all DNA were between 1.8 to 2.0, and the values of AE60/A230were larger than 2. But the yield of DNA extracted by solution I was lower than that of DNA extracted by other solutions. The quality and yield of DNA showed no difference between extraction procedure with liquid nitrogen and without it. All DNA could be used as template to amplify putative prod- ucts with a pair of SPS gene amplified primers and a pair of ISSR primers. All DNA could be digested by different restriction endonuclease. From this study, optimal procedure for isolation of high quality sugarcane genomic DNA was obtained, that is, extraction solution （SDS： 1. 5% ;Tris： 100 mM;EDTA： 20 mM;NaCl： 500 mM）was added to sugarcane leaves after grinding,incubated at 65℃ for 30 minutes, extracted by phenol chloroform and chloroform once ,respectively.
出处
《生物技术通报》
CAS
CSCD
北大核心
2010年第5期101-106,共6页
Biotechnology Bulletin
基金
广西青年科学基金项目(桂科青0991046)
广西甘蔗研究所基本科研业务专项项目(G2009002)
