木瓜ISSR-PCR反应体系的建立与优化  

Establishment and optimization of ISSR-PCR reaction system for Chaenomeles Fructus

作　　者：聂金娥 郭庆梅[2] 王兴军[3] 周凤琴[2] NIE Jin-e;GUO Qing-mei;WANG Xing-jun;ZHOU Feng-qin(Affiliated Hospital of Shandong University of TCM,Jinan 250011,China;Shandong University of TCM,Jinan 250011,China;Shandong Academy of Agricultural Sciences,Jinan 250011,China)

机构地区：[1]山东中医药大学附属医院,山东济南250011 [2]山东中医药大学,山东济南250011 [3]山东省农业科学院,山东济南250011

出　　处：《生物技术》2022年第1期36-40,共5页Biotechnology

基　　金：山东省重点研发计划(2018GSF119004);2019年医疗服务与保障能力提升补助资金(中医药事业传承与发展部分)“全国中药资源普查项目”(财社[2019]39号)。

摘　　要：[目的]获得最佳的木瓜ISSR-PCR扩增反应体系。[方法]采用单因素试验和正交实验对木瓜ISSR-PCR反应体系的DNA模板浓度、引物浓度、延伸时间、循环次数等因素进行梯度优化。[结果]得到了木瓜ISSR-PCR的最佳反应体系(20μL)为引物浓度0.5μmol/L,DNA模板50 ng,Mix 10μL,ddH_(2)O补全;最佳扩增程序为:94℃预变性5 min;94℃变性45 s,退火45 s,72℃延伸90 s,35个循环;72℃延伸7 min,4℃保存。[结论]优化确定的木瓜ISSR-PCR反应体系稳定可靠,为后续木瓜种质资源及遗传多样性研究奠定基础。[Objective]To obstain the most suitable ISSR-PCR system for Chaenomeles Fructus.[Method]Single factor experiment and orthogonal experiment were used to optimize the template DNA,primer concentration,extension time,number of cycles and other factors that affect ISSR-PCR amplification.[Result]Optimum reaction system(20μL)contained primers of 0.5μmol/L,template DNA of 50 ng,10μL of Mix,and ddH_(2)O completed.The optimal amplifying procedure was as follows:pre-denaturing for 5 minutes at 94℃,35 cycles were performed with denaturing of 45 seconds at 94℃,annealing of 45 seconds due to denaturing temperature of different primer,extension of 90 seconds at 72℃,a final extension step of 7 minutes at 72℃and stored at 4℃.[Conclusion]The stable and reproducible optimal ISSR-PCR reaction system was established for Chaenomeles Fructus,which laid a good foundation for germplasm resources identification and genetic diversity study.

关 键 词：木瓜 ISSR-PCR 单因素试验 正交设计 体系优化 

分 类 号：R932[医药卫生—生药学]