永瓣藤ISSR-PCR反应体系的建立与优化  

Establishment and Optimization of ISSR-PCR Reaction System for Monimopetalum chinense

作　　者：郝朝运[1,2] 张小平[1,2] 范睿[1,2] 

机构地区：[1]安徽师范大学安徽省重要生物资源保护与利用研究重点实验室,安徽芜湖241000 [2]安徽师范大学生物环境与生态安全安徽省高校重点实验室,安徽芜湖241000

出　　处：《激光生物学报》2008年第5期701-706,共6页Acta Laser Biology Sinica

基　　金：安徽省自然科学基金项目(050430501);高等学校博士学科点科研基金项目(20060370001);安徽省高校自然科学研究重点资助项目(KJ2007A092)

摘　　要：以永瓣藤基因组DNA为模板,通过单因子、双因子实验研究了ISSR反应体系中主要成分(Mg2+、dNTP、引物、模板DNA、TaqDNA聚合酶)以及热循环参数(退火温度、循环数、变性时间、退火时间、延伸时间)对扩增结果的影响,并找出各自的最适条件,建立了适合永瓣藤ISSR分析的反应体系和扩增程序,即在25μL反应体系中,内含1×PCRbuffer、1.5mmol/LMg2+、200μmol/LdNTP、0.5μmol/L引物、50ng模板、2UTaqDNA聚合酶。扩增程序为94℃预变性5min,然后进行35个循环:94℃变性30s,复性1min,72℃延伸1min,循环结束后72℃延伸7min。这一优化系统的建立为今后利用ISSR标记技术进行永瓣藤鉴定及种质遗传多样性分析提供了一个标准化程序。Based on the genomic DNA extracted from Monimopetalum chinense Rehd, this paper presented the effect of the main reaction system elements （Mg^2＋ , dNTP, primer,template DNA, Taq DNA polymerase） and hot-cycle parameters （annealing temperature ,cycles ,denaturing time,annealing time and extension time） on ISSR-PCR which were tested by single or dual factor experiment respectively, to determine its optimal levels. A reaction system and amplified procedure suitable for M. chinense were established, that is, 25 μL amplification reaction system containing 1 × PCR buffer, 1.5 mmol/L Mg^2＋ , 200 μmol/L dNTP, 0. 5 μmol/L primer, 50 ng template DNA and 2 U TaqDNA polymerase. The optimal amplified procedure was devised for 5 minutes of pre-denaturalization at 94 ℃ , 35 cycles of 30 seconds for denaturalization at 94℃, 60 seconds of annealing due to denaturing temperature of different primer, 60 seconds of extension at 72 ℃, and 7 min of extension at 72 ℃ in the final cycle. This optimal system laid the standardization program of the identification of M. chinense and the efficient use of its germplasm resource.

关 键 词：永瓣藤 ISSR 反应体系 热循环参数 

分 类 号：Q341[生物学—遗传学] Q78