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植物研究 ›› 2009, Vol. 29 ›› Issue (2): 234-241.doi: 10.7525/j.issn.1673-5102.2009.02.019

• 论文 • 上一篇    下一篇

中国木犀科苦丁茶ISSR实验条件优化的研究

郑道君1,2;刘国民1,2*;梁远发3;鄢东海3;令狐昌弟3;田永辉3   

  1. (1.海南大学热带生物资源教育部重点实验室,海口 570228) (2.海南大学苦丁茶研究所,海口 570228) (3.贵州省茶叶科学研究所,湄潭 564100)
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-03-20 发布日期:2009-03-20
  • 通讯作者: 刘国民
  • 基金资助:
     

Studies on Optimization of ISSR Amplified Conditions in Kudingcha Species in Oleaceae of China

ZHENG Dao-Jun;LIU Guo-Min;*;LIANG Yuan-Fa;YAN Dong-Hai;LINGHU Chang-Di;TIANG Yong-Hui   

  1. (1.Key Laboratory of Tropical Biological Researches. MOE,Hainan University,Haikou 570228) (2.Kudingcha Research Institute of Hainan University,Haikou 570228) (3.The Tea Research Institute of Guizhou Province,Meitan 564100)
  • Received:1900-01-01 Revised:1900-01-01 Online:2009-03-20 Published:2009-03-20
  • Contact: LIU Guo-Min
  • Supported by:
     

摘要: 系统地研究了中国木犀科苦丁茶ISSR反应体系中的主要影响因子,建立了一套稳定的ISSRPCR反应参数。筛选出了10个有效引物,并以中国木犀科苦丁茶8个物种共21份种质材料为供试材料对优化后的反应条件的重复性、多态性进行了检测。优化后的反应体系为:10×buffer 2.5 μL,2.0~3.0 mmol·L-1 MgCl2,150~300 μmol·L-1 dNTPs,Taq酶1.0~1.5 U,引物0.4~0.5 μmol·L-1,DNA模板5~320 ng。PCR扩增程序为:94℃预变性4 min,然后按94℃变性40 s,50~54℃退火45 s,72℃延伸120 s,进行35个循环,最后72℃延伸8 min。该反应条件可应用于中国木犀科苦丁茶亲缘关系和遗传多样性分析。

关键词: 木犀科, 苦丁茶, ISSR, 影响因子, 体系优化

Abstract: The influential factors of ISSR for Kudingcha species in Oleaceae of China were systematically studied, a set of stable ISSR-PCR reaction parameters was established. 10 effective ISSR primers were selected out, and with them 21 test germplasm materials from 8 Kudingcha species in Oleaceae of China were examined for the repeatability and polymorphism of the optimized reaction conditions. The optimum ISSR-PCR reaction system was that 2.5 μL 10×PCR buffer, 2.0~3.0 mmol·L-1 MgCl2, 150~300 μmol·L-1 dNTPs, 1.0~1.5 Taq polymerase, 0.4~0.5 μmol·L-1 primers, and 5~320 ng DNA template were contained in 25 μL reaction solution. The optimized amplification program was that pre-denaturing at 94℃ for 4 min, then denaturing at 94℃ for 40 s, primer annealing at 50℃~54℃ for 45 s, extension at 72℃ for 120 s, for 35 cycles, at last extension at 72℃ for 8 min. The productions were stored at 4℃. The optimized ISSR-PCR reaction system was suitable for the study of genetic diversity and relationship of Kudingcha germplasm resources in Oleaceae of China.

Key words: Kudingcha species, Oleaceae, ISSR, influential factors, Optimization of system

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