濒危植物长柄双花木ISSR-PCR反应体系的优化

Optimization of ISSR-PCR reaction system for endangered plant Disanthus cercidifolius var. longipes

采用改进的CTAB法提取长柄双花木（ Disanthus cercidifolius var. longipes）基因组DNA，并以此DNA为模板，对ISSR—PCR的反应体系进行了优化，建立了适合长柄双花木ISSR—PCR的反应体系和程序，即20μL的反应体系中，含模板DNA20ng，Mg2^＋2．0mmol·L^-1，引物0．25μmol·L^-1，dNTPs0．20mmol·L^-1，Taq DNA聚合酶1．0U；扩增程序：94℃预变性5min，94℃变性30S，48—54℃复性45s；72℃延伸90s；共36个循环；循环结束后，72℃延伸7min．本研究为利用这一分子标记对长柄双花木进行遗传多样性分析奠定了基础．

In order to study the genetic diversity of Disanthus cercidifolius var. longipes, an endangered plant in China, improved CTAB method used to extract genomic DNA and this DNA as a template for ISSR-PCR reaction conditions were optimized. The optimal ISSR-PCR system for D. cercidifolius var. longipes was established for the first time, that is, 20 μL of the reaction system, with 2 μL 10 × buffer, 0. 20 mmol · L^-1 dNTP, 0. 25 μmol · L^-1 concentration of primers, 2.0 mmol · L^-1 Mg^2＋ , 1.0 U TaqDNA polymerase, about 20 ng DNA, 14. 1 μL double-distilled water. The optimal amplified procedure was as follows： after a pre-denaturing of 5 min at 94 ℃, followed by 36 cycles which were performed with denaturing of 30 s at 94 ℃, annealing of I rain due to denaturing temperature 48 - 54 ℃ of different primer 45 s, extension 90 s of 1.5 rain at 72 ℃, a final extension step of 7 min at 72℃ and hold at 4 ℃. This paper could settle favorable basis for studying the genetic diversity of D. cercidifolius var. longipes using ISSR molecular marker techniques.