泡桐MSAP体系建立及引物筛选  

Establishment of Paulownia MSAP reaction systems and primers screening

作　　者：曹喜兵[1] 赵改丽[1] 范国强[1] 

机构地区：[1]河南农业大学泡桐研究所,河南郑州450002

出　　处：《河南农业大学学报》2012年第5期535-541,共7页Journal of Henan Agricultural University

基　　金：国家自然科学基金项目(30571496)

摘　　要：以豫杂一号泡桐组培苗为材料,通过对影响MSAP反应体系各主要因素的优化,建立了适于泡桐MSAP分析的反应体系.结果表明,最佳酶切体系(25μL)包含300 ng模板DNA,16 U的EcoR I和10 U的HapⅡ(MspI),各双酶切8 h后,80℃失活20 min;最佳连接体系(25μL)是酶切产物20μL,0.16μmol.L-1EcoR I接头,1.6μmol.L-1HapⅡ(MspI)接头,2.5μL 10×T4Buffer,2 U T4连接酶,22℃连接18 h;最佳预扩反应体系(20μL)是5μL稀释10倍的连接产物,100μmol.L-1dNTP,0.5 U Taq酶,EcoR I和HapⅡ(MspI)预扩引物各0.25μmol.L-1,2.5μL 10×PCR Buffer.最佳选择性扩增反应体系(20μL)为5μL稀释30倍预扩增产物,100μmol.L-1 dNTP,0.5 U Taq酶,EcoR I和HapⅡ(MspI)选扩引物各0.3μmol.L-1,2.5μL 10×PCRBuffer.最后,利用优化的体系,筛选出了96对适宜于泡桐MSAP分析的引物.The establishment of Paulownia MSAP reaction system and its primer screening were inves- tigated through study on the main factors which affected the system quality. The results indicated that the optimum conditions for restriction enzyme digestion of the genomic DNA from the paulownia plant were as follows：300 ng DNA template, 16 U EcoR I and HapⅡ （Msp Ⅰ ）respectively in 25 μL reaction volume at 37℃ for 8 h, and inactivated at 80 ℃ for 20 min;the ligation reaction （25 μL ）with 20 μL digestion product, 0.16μmol·L^-1 EcoR Ⅰadapter, 1.6 μmol·L^-1 HapⅡ （MspⅠ）adapter,2.5 μL 10 × T4 buffer,2 U T4 ligation enzyme at 22 ℃ for 18 h was the optimum condition; the preamplifica-tion reaction （20 IxL） with 5 μL the ligation fragment which was dilutedl0 times, 100 μmol·L^-1 dNTP, 0.5 U Taq enzyme, EcoR I and Hap H （ Msp Ⅰ） primer respectively 0.25 μmol·L^-1,2.5 trL 10× PCR buffer was the optimum condition; the selective amplification reaction （ 20 I^L ） with 5 μL preamplification product which was diluted 30 times, 100 μmol·L^-1 dNTP, 0.5 U Taq enzyme, EcoR I and Hap Ⅱ （Msp Ⅰ ） primer respectively 0. 3 μmol·L^-1 ,2.5 μL 10 × PCR buffer. Finally, by using the optimized system,96 pairs of primers were chosen for the MSAP analysis of paulownia plants.

关 键 词：泡桐 MSAP 反应体系 引物筛选 

分 类 号：S792.43[农业科学—林木遗传育种]