黑老虎ISSR-PCR反应体系的建立与优化  

Establishment and Optimization of ISSR PCR Reaction System for Kadsura coccinea

作　　者：唐健民 漆小雪[1] 蒋运生[1] 熊忠臣[1] 邹蓉[1] TANG Jianmin;QI Xiaoxue;JIANG Yunsheng;XIONG Zhongchen;ZOU Rong(Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization,Guangxi Institute of Botany,Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences,Guilin,Guangxi,541006,China)

机构地区：[1]广西壮族自治区中国科学院广西植物研究所,广西植物功能物质研究与利用重点实验室,广西桂林541006

出　　处：《广西科学》2020年第4期406-412,共7页Guangxi Sciences

基　　金：桂林市科技计划(20160223-1);广西科技攻关项目(桂科AB18221091);中央引导地方科技发展专项(桂科ZY1949013);广西科技基础和人才专项(桂科AD17129022);广西植物功能物质研究与利用重点实验室项目(ZRJJ2018-10)资助。

摘　　要：为建立和优化特色药食两用植物黑老虎(Kadsura coccinea)的ISSR-PCR反应体系和扩增程序,通过正交和单因素试验研究Mg2+、dNTPs、引物、Taq DNA聚合酶、模板DNA、退火温度及循环次数等因素对黑老虎ISSR-PCR反应体系扩增效果的影响。结果表明,在20μL的反应体系中,最佳扩增条件为Mg2+浓度2.5 mmol/L、dNTPs浓度0.1 mmol/L、引物浓度0.6μmol/L、Taq DNA聚合酶用量1.50 U、模板DNA用量45 ng,退火温度50.4℃,循环40次。这一优化体系的建立可为深入开展黑老虎种质资源遗传多样性研究奠定基础。In order to establish and optimize ISSR-PCR reaction system and amplification procedure of the characteristic medicinal and edible Kadsura coccinea,the effects of Mg2+,dNTPs,primers,Taq DNA polymerase,template DNA,annealing temperature and number of cycles on the amplification of ISSR-PCR reaction system of K.coccinea were studied by orthogonal and single-factor experimental design methods.The results showed that in a 20μL reaction system,the optimal reaction system was the system with Mg2+of 2.5 mmol/L,dNTPs of 0.1 mmol/L,primer of 0.6μmol/L,Taq DNA polymerase dosage of 1.50 U and template DNA dosage of 45 ng.The annealing temperature was 50.4℃,and the cycle was 40 times.The establishment of this optimization system can lay a foundation for further research on genetic diversity of K.coccinea germplasm resources.

关 键 词：黑老虎 ISSR-PCR 正交设计 单因素试验 反应体系优化 

分 类 号：R282.7[医药卫生—中药学]