南药益智AFLP-PCR体系的建立与优化

Establishment and optimization of AFLP- PCR system in South- China medicine Alpinia oxyphylla Miquel

[目的]建立适合南药益智的扩增片段长度多态性(AFLP)扩增体系来研究不同地理居群益智遗传多样性。[方法]利用植物基因组试剂盒法提取高质量的益智基因组DNA,采用单因素和正交试验对AFLP过程中的酶切和PCR相关影响因素进行优化,并对适合益智AFLP分析的引物组合进行筛选。[结果]实验结果表明最佳酶切反应体系:模板DNA 0.6μg,酶量20 U,酶切时间2 h;最佳AFLP-PCR选择性扩增反应体系(总体积为25μL):10×PCR Buffer(不含Mg2+)2.5μL,d NTPs 2μL,Mg2+(25mmol/L)1.5μL,引物(10 pmol/μL)各2.0μL,Taq酶(5 U/ml)0.5μL,模板稀释25倍2μL。利用建立的最佳扩增体系从64对引物中筛选获得8对选择性引物适合益智AFLP分析。[结论]建立了稳定的AFLP-PCR体系,为研究益智遗传多样性的AFLP分析奠定了基础。

[Objective]The study intended to establish an AFLP-PCR system and provide technical support to study the genetic diversity of south-China medicine Alpinia oxyphylla Miquel in different geographical populations.[Methods]High quality genomicDNA of Alpinia oxyphylla Miquel was extracted by plant genome kit method.The main factors including enzyme digestion and PCR in the process of AFLP were optimized by single factor and orthogonal experiments,and the primer paris were screened.[Results]The results show that the optical digestion system was templateDNA 0.6μg,amount of enzyme 20 U,the reaction time 2 hours.The optical AFLP-PCR selection amplification system（total volume 25μL） was10 × PCR Buffer（free Mg2 ＋） 2.5μL,d NTPs 2μL,Mg2 ＋（25 mmol /L） 1.5μL,primers（10 pmol/μL） each 2.0μL,TaqDNA polymerase（5 U/ml） 0.5μL,25 times of dilution template 2μL.And 8 pairs of primers were selected from 64 pairs of primer combination for the AFLP-PCR system of Alpinia oxyphylla Miquel.[Conclusion] The stable AFLP-PCR system was established,and can be used in the genetic diversity of for the study of Alpinia oxyphylla Miquel.