海带属配子体DNA提取及ISSR-PCR体系优化  

Genomic DNA Extraction and Optimization of ISSR-PCR Reaction System for Saccharina

作　　者：李言 崔翠菊 罗世菊 李晓捷 赵聚萍 赛珊 钱瑞 武瑞娜 

机构地区：[1]山东东方海洋科技股份有限公司/国家海藻工程技术研究中心/山东省海藻遗传育种与栽培技术重点实验室,山东烟台264003

出　　处：《农学学报》2014年第11期80-84,共5页Journal of Agriculture

基　　金：国家科技支撑计划"海藻栽培新种质苗种繁育与栽培技术研究及产业化示范"(2012BAD55G01);国家863重大项目"基于全基因组信息的藻类遗传选育"(2012AA10A406);烟台市科技发展计划项目"药用和食用海藻新种质苗种繁育与栽培技术研究及产业化示范"(2013LGS002)

摘　　要：为了建立一个重现性好、稳定性高,适用于海带属ISSR遗传分析的PCR反应体系,从海带属配子体材料中提取基因组DNA作为模板,应用正交设计试验对反应体系中各因子的不同浓度进行组合优化。结果显示:适用于海带ISSR扩增反应的最佳体系(20μL)为:1×PCR buffer,1.2 mmol/L Mg2+,0.2 mmol/L d NTPs,1.0μmol/L ISSR引物,40 ng模板DNA,0.25 U Taq DNA聚合酶;扩增PCR程序为:95℃预变性3 min;95℃变性45 s,52℃退火45 s,72℃延伸2 min,共38个循环;最后72℃延伸10 min。在优化的海带ISSR反应条件下,扩增条带稳定,重现性好,为应用ISSR分子标记技术进行海带属遗传多样性研究和分子鉴定奠定了技术基础。In order to establish a reproducible, stable, and suitable reaction system of Saccharina for ISSRanalysis of genetic differences, the genomic DNA extracted from gametophytes of Saccharina was used astemplate, the different concentrations of the factors in reaction system were optimized by orthogonal designexperiment. The results showed that, the optimal ISSR reaction system（20 μL） contained 1 ×PCR buffer, 1.2mmol/L Mg^2＋, 0.2 mmol/L d NTPs, 1 μmol/L ISSR primer, 40 ng template DNA and 0.25 U Taq DNApolymerase. The PCR program was pre-denatured at 95℃ for 3 min and followed by 38 cycles of 45 s at 95℃,45 s at 52℃, 2 min at 72℃, and final extension of 10 min at 72℃. The optimal ISSR reaction system was stableand repeatable. This system could provide the technical foundation for the application of ISSR molecularmarker technology for the study on genetic diversity and molecular identification of Saccharina.

关 键 词：海带属 配子体 DNA提取 ISSR 体系优化 

分 类 号：Q75[生物学—分子生物学]