中国农业科学院农业信息研究所 中国农学会
Agricultural Information Institute of CAAS China Association of Agricultural Science Societies
En
Ch
中国农业科学院农业信息研究所 中国农学会 主办

AI检索

订阅

返回顶部
当前位置: 首页 > 文章 > 苦瓜基因组DNA的提取及ISSR扩增体系的优化 中国农学通报 2013,29 (4) 88-93
Position: Home > Articles > The Extraction of the Genomic DNA in Bitter Gourd and Optimization of ISSR Amplified System Chinese Agricultural Science Bulletin 2013,29 (4) 88-93

苦瓜基因组DNA的提取及ISSR扩增体系的优化

作  者：
田丽波;谷幸幸;商桑;杨衍
单  位：
海南大学热带作物种质资源保护与开发利用教育部重点试验室;中国热带农业科学院作物品种资源研究所
关键词：
苦瓜;基因组DNA;提取方法;ISSR;优化体系
摘  要：
为了快速获取高质量的苦瓜基因组DNA,以便进行苦瓜白粉病抗性基因分子标记研究,比较了不同的DNA提取方法、不同部位的苦瓜叶片提取基因组DNA的产量和质量,探究了苦瓜基因组DNA提取的最佳方法和叶片的最适部位;研究了ISSR-PCR的退火温度,并采用正交设计法对影响ISSR-PCR的Mg2+、dNTPs、Taq酶、模板DNA以及引物浓度等5个因素进行了优化。结果表明,采用改良CTAB法从苦瓜顶端嫩叶中提取的基因组DNAOD260/OD280值在1.8～1.9之间,OD260/OD230值为2.0左右,对DNA样品进行琼脂糖凝胶电泳检测,主带清晰,降解较少,产量和纯度均较高,效果较好。优化后的ISSR-PCR反应体系为:2.5μL10×PCRbuffer,2.5mmol/LMgCl2,250μmol/LdNTPs,10ng模板DNA,0.75UTaq酶,引物浓度0.7μmol/L,反应总体积为25μL,引物UBC826最佳退火温度为53℃。该体系在多次重复中均能获得良好的扩增结果。苦瓜基因组DNA提取的最佳方法为改良CTAB法,最适合的部位为顶端嫩叶。
译  名：
The Extraction of the Genomic DNA in Bitter Gourd and Optimization of ISSR Amplified System
作  者：
Tian Libo 1,2,Gu Xingxing 1,Shang Sang 1,Yang Yan 2 (1 Key Laboratory of Protection and Development Utilization of Tropical Crop Germplasm Resources (Hainan University),Ministry of Education,Haikou 570228;2 Tropical Crops Genetic Resources Institute of Chinese Academy of Tropical Agricultural Science,Danzhou Hainan 571737)
关键词：
bitter gourd;genomic DNA;extraction method;ISSR (inter-simple sequence repeat);optimized system
摘  要：
In order to rapid acquiring high quality genomic DNA of Bitter gourd (Momordica charantia L.) and research the resistance gene molecular marker of powdery mildew (Sphaerotheca fuliginea),the paper compared the extracted genomic DNA ' s yield and quality by different extracted methods and different physiological periods ' leaves of bitter gourd to search the best method and the most suitable period in extracting genomic DNA from bitter gourd.The annealing temperatures of ISSR-PCR (inter-simple sequence repeat and polymerase chain reaction) was researched,and the concentrations of Mg2 +,dNTPs,Taq DNA polymerase,genomic DNA and primers which effect ISSR-PCR were optimized by using orthogonal design method.The result showed that the OD 260 /OD 280 values of the genomic DNA extracted from the top leaves of bitter gourd by improved CTAB method were 1.8-2.0 and the OD 260 /OD 230 values were about 2.0,the electrophoretogram showed that the primary brands were clear and less degradation,the DNA samples were pure and had good quality,the effect was the best.The optimal system was 2.5μL 10×PCR buffer,2.5mmol/L MgCl 2,250 μmol/L dNTPs,10 ng genomic DNA,0.75 U Taq DNA polymerase,0.7μmol/L primers,and the total volume of the reaction was 25μL,the most compatible annealing temperatures of primer UBC826 was 53℃.The optimal system could be employed to gain good results in repeated PCR experiments.The best extracting method was the improved CTAB method and the top tender leaf was the most fitting.

相似文章

导出引用
计量
文章访问数: 8
HTML全文浏览量: 0
PDF下载量: 0

所属期刊

推荐期刊

友情链接Friendly link
农业信息研究所Agricultural information institute 农业智能知识服务平台Agricultural intelligent knowledge service platform 农业学术服务平台Agricultural Knowledge Service System 中国知网CNKI 维普网VIP 万方WANFANG DATA
联系我们Contact us

电话Tell：010-82109652-808

邮箱E-mail：agrijournal@caas.cn

服务Service
关于我们About us 意见建议Opinions and suggest
版权说明Copyright
单位：中国农业科学院农业信息研究所 中国农学会 © 2017中国农业科学院农业信息研究所 ICP备案号：京ICP备09089781号-46