濒危藏药桃儿七cpSSR-PCR引物开发与反应体系优化
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摘要
利用桃儿七叶绿体全基因组开发cpSSR引物,使用正交设计试验,对桃儿七cpSSR-PCR反应体系中的5种主要因素(模板DNA,Mg~(2+),Taq DNA酶,dNTPs及ISSR引物)进行筛选优化来研究桃儿七遗传多样性。选取甘肃碌曲县则岔石林桃儿七居群样本及13个不同桃儿七居群验证该反应体系。正交试验表明:各因素显著性为Mg~(2+)>引物>Taw酶>DNA浓度>dNTPs,桃儿七cuss-PCR反应的最佳体系为(25μL):DNA20 ng/μL,Mg~(2+)1.5 mmol/L,dNTPs 2.0 mmol/L,Taq DNA酶0.5 U,引物1.0μmol/L,2.5μL 10×Buffer。该体系具有很高的稳定性和重复性,并为野生桃儿七遗传多样性及保护策略研究提供了技术支持。
The cpSSR primer was developed by the whole chloroplast genome of Sinopodophyllum hexandrum.The five main factors(template DNA, Mg~(2+), Taq DNA polymerase, dNTPs and ISSR primer) in the cpSSR-PCR reaction system of S. hexandrum were screened and optimized by orthogonal design test to study the genetic diversity of S. hexandrum. The population sample of S. hexandrum in Shilin, Zecha, Luqu County, Gannan, Gansu Province and 13 different S. hexandrum populations were selected to verify the reaction of system. The result of orthogonal design test showed that the significance of each factor was Mg2 +> primer > Taq polymerase > DNA template > dNTPs. The best system for cuss-PCR reaction(25 μL) in S. hexandrum was 20 ng/μL DNA, 1.5 mmol/L Mg~(2+), 0.2 mol/L dNTP, 0.5 U Taq polymerase, 1.0 μmol/L primer, and 2.5 μL 10× Buffer. The system had high stability and repeatability, and it could provide technical support for the genetic diversity and protection strategy of wild S. hexandrum.
The cpSSR primer was developed by the whole chloroplast genome of Sinopodophyllum hexandrum.The five main factors(template DNA, Mg~(2+), Taq DNA polymerase, dNTPs and ISSR primer) in the cpSSR-PCR reaction system of S. hexandrum were screened and optimized by orthogonal design test to study the genetic diversity of S. hexandrum. The population sample of S. hexandrum in Shilin, Zecha, Luqu County, Gannan, Gansu Province and 13 different S. hexandrum populations were selected to verify the reaction of system. The result of orthogonal design test showed that the significance of each factor was Mg2 +> primer > Taq polymerase > DNA template > dNTPs. The best system for cuss-PCR reaction(25 μL) in S. hexandrum was 20 ng/μL DNA, 1.5 mmol/L Mg~(2+), 0.2 mol/L dNTP, 0.5 U Taq polymerase, 1.0 μmol/L primer, and 2.5 μL 10× Buffer. The system had high stability and repeatability, and it could provide technical support for the genetic diversity and protection strategy of wild S. hexandrum.
引文
Bao L.Y.,Yang X.L.,and Liu Y.J.,2004,The biological properties of sinopodophyllum hexandrum and its planting technology,Zhongguo Linfutechan(Forest By-Product and Speciality in China),(4):1-2(鲍隆友,杨小林,刘玉军,2004西藏野生桃儿七生物学特性及人工栽培技术研究,中国林副特产,(4):1-2)
Deng K.J.,Zhang Y.,Xiong B.Q.,Peng J.H.,Zhang T.,and Reng Z.L.,2009,Identification,characterization and utilization of simple sequence repeat markers derived from salvia miltiorrhiza expressed sequence tags,Yaoxue Xuebao(Acta Phytotaxon Sinica),44(10):1165-1172(邓科君,张勇,熊丙全彭金华,张韬,赵晓楠,任正隆,2009,药用植物丹参EST-SSR标记的鉴定,药学学报,44(10):1165-1172)
Dong J.Z.,Yi Z.L.,and Jiang J.X.,2005,Current situation of the research on the germplasm resources of medicinal plants in China,Xibu Linye Kexue(Journal of West China Forestry Science),34(2):95-101(董静洲,易自力,蒋建雄,2005,我国药用植物种质资源研究现状,西部林业科学,34(2)95-101)
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Jiang F.J.,Tang D.C.,and Tang N.,2017,Construction and optimization of cp DNA-PCR system for Lilium pumilum DC.Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(1):213-222(蒋福娟,唐道城,唐楠,2017,山丹百合cp DNA-PCR反应体系建立与优化,分子植物育种,15(1):213-222)
Jiang Z.G.,and Ma K.P.,2017,The state's will,scientific decision and citizen participation:in memory of the first provincial species red list in China,Shengwu Duoyangxing(Biodiversity Science),25(7):794-795(蒋志刚,马克平,2017,中国生物多样性保护的国家意志,科学决策和公众参与:第一份省域物种红色名录研究,生物多样性,25(7):794-795)
Lalitha S.,2000,Primer premier 5,Biotech Software and Internet Report.,1(6):270-272
Li M.F.,and Zheng X.Q.,2004,Research progress of methods of SSRprimers development,Yichuan(Hereditas),26(5):769-777(李明芳,郑学勤,2004,开发SSR引物方法之研究动态,遗传,26(5):769-776)
Li N.,Jiao Z.,and Qin G.Y.,2005,DNA molecular maker techniques and their application in wheat breadding and genetic study,Henongxue Bao(Journal of Nuclear Agricultural Sciences),19(4):322-326(李娜,焦浈,秦广雍,2005,DNA分子标记技术及其在小麦育种及遗传研究中的应用,核农学报,19(4):322-326)
Li Q.,and Wan J.M.,2005,SSRHunter:Development of a local searching software for SSR sites,Yichuan(Hereditas),3(5):808-810(李强,万建民,2005,SSRHunter:一个本地化的SSR位点搜索软件的开发遗传,3(5):808-810)
Liu W.J.,Xu J.,Yin S.L.,Tian Y.,and Li J.S.,2017,Mechanisms of benefit-sharing of medicinal plants found in China and neighboring countries,Shengwu Duoyangxing(Biodiversity Science),25(8):907-913(刘文静,徐靖,银森录,田瑜,李俊生,2017,探讨我国与邻国共有药用植物的惠益分享机制,生物多样性,25(8):907-913)
Liu W.,Yin D.,Liu J.,and Li N.,2014,Genetic diversity and the qinling mountains,China,PLo S One,9(10):e110500
Martins W.S.,Lucas D.C.S.,Neves K.F.S.,and Bertioli D.J.,2009,Web Sat-A web software for microsatellite marker development,Bioinformation,3(6):282-283
Meng L.,Liu R.,Chen J.,and Ding C.,2017,The complete chloroplast genome of Sinopodophyllum hexandrum Ying(B-erberidaceae),Mitochondrial DNA A DNA Mapp.Seq.Anal.,28(3):342-343
Moore S.S.,Sargeant L.L.,King T.J.,Mattick J.S.,Georges M.,and Hetzel D.J.S.,1991,The conservation of dinucleotide microsatellites among mammalian genomes allows the use of heterologous PCR primer pairs in closely related species,Genomics,10(3):654-660
Rychlik W.,2007,OLIGO 7 primer analysis software,PCRPrimer Design,402:35-59
Sharma K.D.,Singh B.M.,Sharma T.R.,Katoch M.,and Guleria S.,2000,Molecular analysis of variability in podophyllum hexandrum royle-an endangered medicinal herb of northwestern himalaya,Plant Genetic Resources Newsletter,124:57-61
Xiao M.,Li Q.,Guo L.,Tang L.,Wang L.,and Chen F.,2015,RAPD analysis of genetic diversity of an endangered species Sinopodophyllum hexandrum(Royle)Ying from western Sichuan province,China,Shengtai Xuebao(Acta Ecologica Sinica),35(5):1488-1495(肖猛,李群,郭亮,唐琳,王丽,陈放,2015,四川西部濒危植物桃儿七遗传多样性的RAPD分析,生态学报,35(5):1488-1495)
Xu Z.A.,Wang T.B.,Li C.Y.,Bao L.Y.,Ma Q.M.,and Miao Y.L.2002,Brief Introduction to the orthogonal test design,Kej Qingbao Kaifa Yu Jingji(Sci/Tech Information Developmen and Ecolomy),12(5):148-150(徐仲安,王天保,李常英,暴丽艳,马青梅,苗玉宁,2002,正交试验设计法简介,科技情报开发与经济,12(5):148-150)
Xue D.Y.,2011,Analysis for the main elements and potential impacts of nagoya protocol,Shengwu Duoyangxing(Biodiversity Science),19(1):113-119(薛达元,2011,《名古屋议定书》的主要内容及其潜在影响,生物多样性,19(1):113-119)
Zhang D.,Ren M.Y.,Guan X.,and Zhang Y.D.,2017,Establishment and optimization of ISSR-PCR reaction system for Sinopodophyllum hexandrum(Royle)Ying,Fenzi Zhiwu Y-uzhong(Molecular Plant Breeding),15(4):1408-1416(张盾,任梦云,关潇,张银东,2017,濒危藏药桃儿七ISSR-PCR反应体系的建立与优化,分子植物育种,15(4):1408-1416)
Deng K.J.,Zhang Y.,Xiong B.Q.,Peng J.H.,Zhang T.,and Reng Z.L.,2009,Identification,characterization and utilization of simple sequence repeat markers derived from salvia miltiorrhiza expressed sequence tags,Yaoxue Xuebao(Acta Phytotaxon Sinica),44(10):1165-1172(邓科君,张勇,熊丙全彭金华,张韬,赵晓楠,任正隆,2009,药用植物丹参EST-SSR标记的鉴定,药学学报,44(10):1165-1172)
Dong J.Z.,Yi Z.L.,and Jiang J.X.,2005,Current situation of the research on the germplasm resources of medicinal plants in China,Xibu Linye Kexue(Journal of West China Forestry Science),34(2):95-101(董静洲,易自力,蒋建雄,2005,我国药用植物种质资源研究现状,西部林业科学,34(2)95-101)
Fu L.G.,ed.,1992,plant red data book-The rare and endangered plants in China,Science Press,Beiing,China,pp.184(傅立国,1992,中国植物红皮书-稀有濒危植物,科学出版社中国,北京,pp.184)
Jiang F.J.,Tang D.C.,and Tang N.,2017,Construction and optimization of cp DNA-PCR system for Lilium pumilum DC.Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(1):213-222(蒋福娟,唐道城,唐楠,2017,山丹百合cp DNA-PCR反应体系建立与优化,分子植物育种,15(1):213-222)
Jiang Z.G.,and Ma K.P.,2017,The state's will,scientific decision and citizen participation:in memory of the first provincial species red list in China,Shengwu Duoyangxing(Biodiversity Science),25(7):794-795(蒋志刚,马克平,2017,中国生物多样性保护的国家意志,科学决策和公众参与:第一份省域物种红色名录研究,生物多样性,25(7):794-795)
Lalitha S.,2000,Primer premier 5,Biotech Software and Internet Report.,1(6):270-272
Li M.F.,and Zheng X.Q.,2004,Research progress of methods of SSRprimers development,Yichuan(Hereditas),26(5):769-777(李明芳,郑学勤,2004,开发SSR引物方法之研究动态,遗传,26(5):769-776)
Li N.,Jiao Z.,and Qin G.Y.,2005,DNA molecular maker techniques and their application in wheat breadding and genetic study,Henongxue Bao(Journal of Nuclear Agricultural Sciences),19(4):322-326(李娜,焦浈,秦广雍,2005,DNA分子标记技术及其在小麦育种及遗传研究中的应用,核农学报,19(4):322-326)
Li Q.,and Wan J.M.,2005,SSRHunter:Development of a local searching software for SSR sites,Yichuan(Hereditas),3(5):808-810(李强,万建民,2005,SSRHunter:一个本地化的SSR位点搜索软件的开发遗传,3(5):808-810)
Liu W.J.,Xu J.,Yin S.L.,Tian Y.,and Li J.S.,2017,Mechanisms of benefit-sharing of medicinal plants found in China and neighboring countries,Shengwu Duoyangxing(Biodiversity Science),25(8):907-913(刘文静,徐靖,银森录,田瑜,李俊生,2017,探讨我国与邻国共有药用植物的惠益分享机制,生物多样性,25(8):907-913)
Liu W.,Yin D.,Liu J.,and Li N.,2014,Genetic diversity and the qinling mountains,China,PLo S One,9(10):e110500
Martins W.S.,Lucas D.C.S.,Neves K.F.S.,and Bertioli D.J.,2009,Web Sat-A web software for microsatellite marker development,Bioinformation,3(6):282-283
Meng L.,Liu R.,Chen J.,and Ding C.,2017,The complete chloroplast genome of Sinopodophyllum hexandrum Ying(B-erberidaceae),Mitochondrial DNA A DNA Mapp.Seq.Anal.,28(3):342-343
Moore S.S.,Sargeant L.L.,King T.J.,Mattick J.S.,Georges M.,and Hetzel D.J.S.,1991,The conservation of dinucleotide microsatellites among mammalian genomes allows the use of heterologous PCR primer pairs in closely related species,Genomics,10(3):654-660
Rychlik W.,2007,OLIGO 7 primer analysis software,PCRPrimer Design,402:35-59
Sharma K.D.,Singh B.M.,Sharma T.R.,Katoch M.,and Guleria S.,2000,Molecular analysis of variability in podophyllum hexandrum royle-an endangered medicinal herb of northwestern himalaya,Plant Genetic Resources Newsletter,124:57-61
Xiao M.,Li Q.,Guo L.,Tang L.,Wang L.,and Chen F.,2015,RAPD analysis of genetic diversity of an endangered species Sinopodophyllum hexandrum(Royle)Ying from western Sichuan province,China,Shengtai Xuebao(Acta Ecologica Sinica),35(5):1488-1495(肖猛,李群,郭亮,唐琳,王丽,陈放,2015,四川西部濒危植物桃儿七遗传多样性的RAPD分析,生态学报,35(5):1488-1495)
Xu Z.A.,Wang T.B.,Li C.Y.,Bao L.Y.,Ma Q.M.,and Miao Y.L.2002,Brief Introduction to the orthogonal test design,Kej Qingbao Kaifa Yu Jingji(Sci/Tech Information Developmen and Ecolomy),12(5):148-150(徐仲安,王天保,李常英,暴丽艳,马青梅,苗玉宁,2002,正交试验设计法简介,科技情报开发与经济,12(5):148-150)
Xue D.Y.,2011,Analysis for the main elements and potential impacts of nagoya protocol,Shengwu Duoyangxing(Biodiversity Science),19(1):113-119(薛达元,2011,《名古屋议定书》的主要内容及其潜在影响,生物多样性,19(1):113-119)
Zhang D.,Ren M.Y.,Guan X.,and Zhang Y.D.,2017,Establishment and optimization of ISSR-PCR reaction system for Sinopodophyllum hexandrum(Royle)Ying,Fenzi Zhiwu Y-uzhong(Molecular Plant Breeding),15(4):1408-1416(张盾,任梦云,关潇,张银东,2017,濒危藏药桃儿七ISSR-PCR反应体系的建立与优化,分子植物育种,15(4):1408-1416)