鳞尾木ISSR-PCR反应体系的建立与正交优化  

Establishment and Orthogonal Optimization of ISSR-PCR Reaction System of Champ ere ia manillana

作　　者：朱成豪 唐健民 韦记青[2] 高丽梅[2] 邹蓉[2] 秦惠珍 Zhu Chenghao;Tang Jianmin;Wei Jiqing;Gao Limei;Zou Rong;Qin Huizhen(School of Pharmacy,Gulin Medial Universily,Guilin,541004;Guangxi Institute of Botany Chinese Academy of Sciencs,Guilin,541006)

机构地区：[1]桂林医学院药学院,桂林541004 [2]广西壮族自治区中国科学院广西植物研究所,桂林541006

出　　处：《分子植物育种》2020年第22期7476-7484,共9页Molecular Plant Breeding

基　　金：广西植物功能物质研究与利用重点实验室自主研究课题(ZRJJ20189);中央引导地方科技发展专项资金(桂科ZY1949013);广西科技基础和人才专项(桂科AD17129022);广西植物研究所基本业务经费(桂植业18013,18014,19002)共同资助.

摘　　要：为建立与优化广西药食两用植物鳞尾木的ISSR-PCR反应体系和扩增程序,本研究采用正交和单因素试验设计方法,对影响鳞尾木ISSR-PCR反应体系的d NTPs、Taq DNA聚合酶、引物、模板DNA及Mg2+浓度5个因素进行了优化试验,建立了稳定的、可重复的鳞尾木ISSR-PCR扩增反应体系。结果显示:在20μL的反应体系中,dNTPs浓度为0.4 mmol/L、Taq DNA聚合酶用量为1.5 U、引物浓度为0.8μmol/L、Mg2+浓度为2.0 mmol/L、模板DNA浓度为45 ng。扩增程序为:在94℃条件下预变性5 min;94℃条件下变性30 s,52.9℃条件下退火30 s,72℃条件下延伸30 s,以上3个步骤循环40次,最后72℃延伸10 min。这一优化体系的建立可为深入开展鳞尾木种质资源遗传多样性的研究提供帮助。The purpose of this study was to establish and optimize the ISSR reaction system and amplification procedure for the Champereia manillana. The orthogonal and single factor experimental design methods were used to optimize the effects of dNTPs, Taq DNA polymerase, primers, template DNA and Mg2+on the ISSR-PCR reaction system of Champereia manillana. A stable and reproducible ISSR-PCR amplification reaction system was established. The test results were as follows: in a 20 μL reaction system, the dNTPs concentration was 0.4 mmol/L,the Taq DNA polymerase amount was 1.5 U, the primer concentration was 0.8 μmol/L, the Mg2+concentration was 2.0 mmol/L, and the DNA concentration was 45 ng. The amplification procedure was as follows: predenaturation for 5 min at 94℃;denaturation for 30 s at 94℃, annealing for 30 s at 52.9℃, extension for 30 s at 72℃, 40 cycles of the above three steps, and extension for 10 min at 72℃. The establishment of this optimization system could be helpful for further research on the genetic diversity of the Champereia manillana.

关 键 词：鳞尾木(Champereia manillana) ISSR-PCR反应体系 正交试验 

分 类 号：S647[农业科学—蔬菜学]