部分柿属植物IRAP反应体系的建立和指纹图谱构建  

Establishment of the Inter-retrotransposon Amplified Polymorphism(IRAP) Reaction System and Construction of Fingerprint in Some Diospyros spp.

作　　者：杜晓云[1] 罗正荣[1] 

机构地区：[1]华中农业大学园艺植物生物学教育部重点实验室,武汉430070

出　　处：《农业生物技术学报》2006年第6期931-936,共6页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.30471203)资助。

摘　　要：为建立柿属(Diospyros)植物反转录转座子间扩增多态性(IRAP)技术体系,对IRAP分析中影响PCR扩增结果的若干因子进行了研究。确立了适合柿属植物IRAP分析的技术体系:20μL反应体系中,1×Buffer、模板DNA50ng、Mg2+2.0mmol/L、dNTPs0.25mmol/L、引物0.40μmol/L、TaqDNA聚合酶1.0U,矿物油20μL;PCR扩增程序:95℃5min;95℃1min,56℃1min,升温速率+0.6℃/s,72℃2min,循环44次;72℃延伸8min。该优化体系在7种柿属植物33个基因型的I-RAP分析中获得较理想的扩增结果,扩增片段120～1500bp,不同种和柿种以下不同品种间扩增条带总数和带谱相同。每个基因型都有其独特的指纹图谱,可作为区分不同基因型的依据,尤其芽变品种的鉴别。Several main factors influencing the IRAP-PCR reaction were analyzed in order to establish the inter-retrotransposon amplified polymorphism （IRAP） molecular marker system in Diospyros spp.. The optimal system was as follows （in 20 μL reaction system）： Buffer Ix, template DNA 50 ng, Mg^2＋ 2.0 mmol/ L, dNTPs 0.25 mmol/ L, primer 0.40 μmol/ L, Taq DNA polymerase 1.0 U and mineral oil 20 μL. The optimal amplification program was： 1 cycle at 95 ℃, 5 min; 1 cycle at 95 ℃, 1 min; 56 ℃ 1 min; ramp ＋0.6℃/s to 72 ℃ ; 44 cycles of 72 ℃, 2 min, 1 cycle at 72 ℃, 8 min. Steadily and realibly profile was obtained in the 33 genotypes belonging to 7 species using the optimal system and amplification program. The size of fragments obtained ranged from 120-1500 bp, the total bands and amplification pattern were distinctive in different species and cultivals. Every genotype produced an unique fingerprint according to which different genotypes could be distinguished from each other, especially between bud spots.

关 键 词：反转录转座子 IRAP-PCR 技术体系 指纹图谱 

分 类 号：S188[农业科学—农业基础科学]