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生态学杂志 ›› 2009, Vol. 28 ›› Issue (12): 2444-2451.

• 研究报告 • 上一篇    下一篇

濒危药用植物厚朴ISSR引物筛选及反应条件的优化

于华会1;杨志玲1**;杨旭1;谭梓峰1;舒枭2   

  1. 1中国林业科学研究院亚热带林业研究所, 浙江富阳311400|2云南农业大学,昆明 650201
  • 出版日期:2009-12-10 发布日期:2009-12-10

Primer screening and optimization of ISSR reaction condition for endangered medicinal plant |Magnolia officinalis.

YU Hua-hui1|YANG Zhi-ling 1|YANG Xu1;TAN Zi-feng1|SHU Xiao2   

  1. 1Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Fuyang 311400, Zhejiang, China|2Yunnan Agricultural University, Kunming 650201, China
  • Online:2009-12-10 Published:2009-12-10

摘要: 以厚朴DNA为模板,利用正交试验分别对影响厚朴ISSR-PCR反应的Taq 酶浓度、dNTP浓度、引物浓度、Mg2+浓度、模板DNA浓度进行了优化,并通过梯度PCR确定不同引物的最佳退火温度和循环次数,最终确定厚朴最佳反应体系及扩增条件为:25 μl体系,其中包括1.5 mmol·L-1MgCl2,0.3 μmol·L-1引物,0.04 U·μl-1Taq酶,0.2 mmol·L-1 dNTP,4 ng·μl-1 模板DNA,1×Buffer;扩增程序:94 ℃预变性5 min,94 ℃变性30 s,50 ℃~60 ℃(退火温度随引物不同而定)退火45 s,72 ℃延伸90 s,共40个循环,然后72 ℃延伸8 min,4 ℃终止反应。此外,还利用优化的反应体系成功筛选出21条ISSR引物,并利用部分引物对厚朴个体进行了遗传多样性分析。

关键词: 热值, 灰分, 月变化, 棕榈植物

Abstract: To optimize the inter simple sequence repeat (ISSR) reaction condition for Magnolia officinalis genomic DNA, the concentrations of Mg2+, Taq polymerase, primers, deoxyribonucleot-ide triphosphate (dNTP), and template DNA were studied with an orthogonal experimental design, and the optimal anneal temperature of different primers and cycles were determined through gradient PCR. The optimal PCR system for ISSR analysis was 1.5 mmol MgCl2·L-1, 03 μmol primer·L-1 0.04 U Taq polymerase·μl-1, 0.2 mmol dNTP·L-1, 4 ng DNA template·μl-1, and 1×Buffer in 25 μl reaction solution, and the augmentation procedre was predenaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing at 50 ℃-60 ℃ for 45 s, extension at 72 ℃ for 90 s, reaction with 40 cycles, and extension at 72 ℃ for 8 min. Twentyone ISSR primers were screened by using the optimal reaction system, and the genetic diversity was analyzed with some primers.

Key words: Caloric value, Ash content, Monthly changes, Palmae species