海南黎药胆木PsbA-trnH-PCR体系的建立与优化
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摘要
为了建立适合黎药胆木的PsbA-trnH-PCR体系来研究不同地理居群胆木遗传多样性,本研究以植物基因组试剂盒法提取胆木基因组DNA为模板,采用单因素实验和正交试验对PsbA-trnH-PCR过程中的关键影响因素进行优化,并对PsbA-trnH-PCR产物进行测序鉴定。结果表明,最佳PsbA-trnH-PCR反应体系(25μL)为:Taq酶1.0 U,dNTPs 0.4 mmol/L,Mg~(2+)0.75 mmol/L,引物0.15μmol/L,模板20 ng,10×PCR Buffer(不含Mg~(2+))2.5μL;采用该最佳体系对胆木基因组DNA进行PCR扩增,获得扩增产物,经单向测序获得了胆木PsbA-trnH部分序列;并建立了稳定的PsbA-trnH-PCR体系,为胆木的药材鉴别及其遗传多样性研究奠定了基础。
The study intended to establish a PsbA-trnH-PCR system and provide technical support to study the genetic diversity of Li Nationality medicine Nauclea officinalis Pierre ex Pitard in different geographical populations. High quality genomic DNA of N. officinalis Pierre ex Pitard was extracted by the plant genome kit method. The main factors in the process of PsbA-trnH-PCR were optimized by single factor and orthogonal design experiments, and PsbA-trnH-PCR products were sequenced. The results showed that the optimal PsbA-trnH-PCR reaction system(25 μL) was Taq DNA polymerase 1.0 U, dNTPs 0.4 mmol/L, Mg~(2+)0.75 mmol/L, primers 0.15 μmol/L,template DNA 10 ng, 10×PCR Buffer(free Mg~(2+)) 2.5 μL. The genomic DNA of N. officinalis was amplified using the optimal PsbA-trnH-PCR system to obtain the amplification products, which were sequenced to obtain Psb A-trn H partial sequences. The stable PsbA-trnH-PCR system was established, and can be used in the identification and genetic diversity of for the study of N. officinalis.
The study intended to establish a PsbA-trnH-PCR system and provide technical support to study the genetic diversity of Li Nationality medicine Nauclea officinalis Pierre ex Pitard in different geographical populations. High quality genomic DNA of N. officinalis Pierre ex Pitard was extracted by the plant genome kit method. The main factors in the process of PsbA-trnH-PCR were optimized by single factor and orthogonal design experiments, and PsbA-trnH-PCR products were sequenced. The results showed that the optimal PsbA-trnH-PCR reaction system(25 μL) was Taq DNA polymerase 1.0 U, dNTPs 0.4 mmol/L, Mg~(2+)0.75 mmol/L, primers 0.15 μmol/L,template DNA 10 ng, 10×PCR Buffer(free Mg~(2+)) 2.5 μL. The genomic DNA of N. officinalis was amplified using the optimal PsbA-trnH-PCR system to obtain the amplification products, which were sequenced to obtain Psb A-trn H partial sequences. The stable PsbA-trnH-PCR system was established, and can be used in the identification and genetic diversity of for the study of N. officinalis.
引文
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[14]王和飞,张燕,林应邀,等.胆木组织培养与快速繁殖技术研究[J].热带作物学报,2011,32(10):1 878-1 882.
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[2]刘明生.黎药学概论[M].北京:人民卫生出版社,2008:156.
[3]CBOL Plant Working Group.A DNA barcode for land plants[J].Proceedings of the National Academy of Sciences of the United States of America,2009,106(31):12 794-12 797.
[4]夏至,冯翠元,高致明,等.黄芩及其同属近缘种的DNA条形码鉴定研究[J].中草药,2014,45(1):107-112.
[5]陈倩,刘义梅,刘震,等.ITS2和Psb A-trn H序列鉴别马鞭草科药用植物[J].中国中药杂志,2012,37(8):1 107-1 113.
[6]刘涛,纪运恒.蒿属药用植物叶绿体上的psb A-trn H序列分析[J].中国农学通报,2009,25(12):46-49.
[7]张雅琴,石钺,宋明,等.蕨类药材石韦及其混伪品的psb A-trn H序列鉴定[J].中国中药杂志,2014,39(12):2 222-2 226.
[8]Peter M,Hollingsworth,Sean W,et al.Choosing and using a plant DNA barcode[J].Plo S ONE,2011,6:e19254
[9]Kress W J,Erickson D L.A two-locus global DNA barcode for land plants:The coding rbc L gene complements the noncoding psb A-trn H apacer region[J].Plo S ONE,2007,2:e508.
[10]Yao H,Song J Y,Ma X Y,et al.Identification of dendrobium species by a candidate DNA barcode sequence:The chloroplast psb A-trn H intergenic region[J].Planta Medica,2009,75(6):667-669.
[11]Song J Y,Yao H,Li Y,et al.Authentication of the family polygonaceae in Chinese pharmacopoeia by DNA barcoding technique[J].Journal of Ethnopharmacology,2009,124(3):434-439.
[12]Sook Y L,Kooi Y K,Vikneswaran M,et al.Natural indole butyrylcholinesterase inhibitors from Nauclea officinalis[J].Phytomedicine International Journal of Phytotherapy&Phytopharmacology,2015,22(1):45-48.
[13]Li N,Cao L,Cheng Y,et al.In vivo anti-inflammatory and analgesic activities of strictosamide from Nauclea officinalis[J].Pharmaceutical Biology,2014,52(11):1 445-1 450.
[14]王和飞,张燕,林应邀,等.胆木组织培养与快速繁殖技术研究[J].热带作物学报,2011,32(10):1 878-1 882.
[15]杨卫丽,刘明生,毛彩霓,等.胆木的生药学研究[J].中药材,2008,31(9):1 324-1 325.
[16]Shaw J,Lickey E B,Beck J T,et al.The tortoise and the hare II:Relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis[J].Americam Journal of Botanity,2005,92(1):142-166.
[17]夏至,王璐静,李贺敏,等.基于psb A-trn H基因序列对中药材茜草的分子鉴定[J].时珍国医国药,2016,27(2):374-376.
[18]白明明,孙小芹,郭建林,等.基于psb A-trn H分析的何首乌野生居群遗传多样性[J].植物资源与环境学报,2012,21(2):36-44.