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中国农学通报

中国农学通报 ›› 2014, Vol. 30 ›› Issue (16): 164-169.doi: 10.11924/j.issn.1000-6850.2013-2921

• 林学 园艺 园林 • 上一篇    下一篇

广东紫珠ISSR-PCR反应体系建立及引物筛选

房海灵 卢艳花 聂韡 朱培林   

  • 收稿日期:2013-11-07 修回日期:2014-02-11 出版日期:2014-06-05 发布日期:2014-06-05
  • 基金资助:
    江西省科技支撑计划项目“广东紫珠种质资源质量评价及优良种质筛选研究”(20111BBG70030-4)；江西省博士后科研择优资助项目“广东紫珠种质资源多样性评价及药材品质形成的影响机制研究”；江西省财政林业专项“栀子、枳壳、广东紫珠等特色森林药材良种选育利用”(2011511201)；国家青年科学基金项目“基于谱效关系模型的广东紫珠生长及加工过程中质量动态控制研究”(81102799)。

Optinization of ISSR-PCR reaction system and primers screening in Callicarpa kwangtungensis

  • Received:2013-11-07 Revised:2014-02-11 Online:2014-06-05 Published:2014-06-05

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摘要： 为建立稳定性、重复性好的广东紫珠ISSR-PCR反应体系，以广东紫珠总DNA为试验材料，通过单因子结合正交试验对模板DNA浓度、dNTPs浓度、Mg2+浓度、引物浓度和Taq酶用量进行优化，确立了适用于广东紫珠的最佳ISSR-PCR反应体系：0.25 mmol/LdNTPs、1.00 U Taq DNA聚合酶、0.75μmol/L引物、2.0 mmol/L Mg2+、100 ng模板DNA，总反应体积为25μL。同时建立重复性和稳定性均较好的ISSR-PCR扩增程序：预变性94℃，5 min；变性94℃，30 s；退火温度与每个引物相对应，退火45 s；72℃延伸90 s；34个循环；后72℃延伸10 min；4℃保存，扩增结束。用来自不同居群4个个体，以100个ISSR引物进行PCR扩增，筛选出扩增效果较好的20个引物。

关键词: 中介蝮蛇, 中介蝮蛇, 纤溶酶, 原核表达, 载体构建

Abstract: In order to establish a stable, repeatable ISSR-PCR reaction system of Callicarpa kwangtungensis, the factors included the concentration of DNA template, dNTPs, Mg2+, primers and TaqDNA polymerase were studied through the single factor tests, orthogonal tests with the total DNA of C. kwangtungensis as experimental material. And the best reaction system of ISSR-PCR to C. kwangtungensis was established as follows: 0.25 mmol/L dNTPs, 1.00 U TaqDNA polymerase, 0.75μmol/L primers, 2.0 mmol/L Mg2+, 100 ng template DNA in 25μL reaction volume. PCR amplification program of better repeatability and stability was set up after a pre- degeneration 5 min at 94℃; degeneration 30 s at 94℃; annealing time 45 s, annealing temperature corresponds to each primer; extension 90 s at 72℃; 34 cycle from degeneration to extension in total; then extension 10 min at 72℃, finally conserved at 4℃. One hundred ISSR primers were used to screen the suitable primers with 4 samples from different populations of C. kwangtungensis, and 20 ISSR primers with good amplification were screened out.