膜荚黄芪亲缘关系RAPD分析及有效成分的比较研究
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摘要
本研究以16份膜荚黄芪为材料,应用RAPD分子标记技术,进行膜荚黄芪的亲缘关系研究。同时,本研究采用紫外分光光度法测定多糖、总皂苷及黄酮含量,根据化学成分含量分析结果,对不同产地的膜荚黄芪种质资源进行评价。目的是为膜荚黄芪的杂交育种、种质改良和合理栽培提供理论基础。本研究的主要结果如下:
1.通过模板DNA、引物、dNTP、MgCl_2及Taq DNA聚合酶浓度对RAPD反应探索建立了膜荚黄芪RAPD反应优化体系。即在20μL反应体积中,模板DNA 5ng,引物0.4μmol/L,dNTP 150μmol/L,MgCl_2 2.0 mmol/L,Taq DNA聚合酶0.5 unit,1×Buffer;反应程序为预变性94℃5min,94℃45s、36℃60s、72℃90s共循环40次,最后72℃延伸7min。
2.应用RAPD技术对16份膜荚黄芪种质资源进行分析,从200条随机引物中筛选出12条引物,共扩增出148条带,其中127条为多态带,多态性比率为85.8%,多态性比率较高。对16份膜荚黄芪不同种源进行RAPD扩增,根据遗传距离进行聚类分析结果,将其为3类:第一类为野生黄芪;第二类为华北黄芪,包括山西辉远和内蒙古包头;大部分属于第三类,为东北黄芪。
3.不同种质的有效成分含量差异极显著。野生黄芪中多糖、总皂苷及黄酮含量显著高于所有栽培黄芪;栽培黄芪中,引自吉林通化膜荚黄芪多糖含量最高,但总皂苷和黄酮含量较低;引自吉林白山膜荚黄芪虽然多糖含量较低,但皂苷和黄酮含量在栽培黄芪中最高。因此,需根据不同栽培目的选用不同的种质作为繁殖材料。
RAPD analysis was used to evaluate genetic relationship for 16 accessions germplasm resources of Astragalus membranaceus,collected from abroad and different areas in our country.And effective component analysis was carried out to screen the highest germplsm of effective component for them with UV spectrophotometric method.The aim was to establish the foundation for A.membranaceus breeding and genetic improvement.The main results were summarized as follows.
1.Optimization of RAPD Reaction System for A membranaceus was founded.That was in a 20-ml volume,5 ng template DNA,0.4μmol/L primer,150μmol/L dNTP,2.0 mmol/L MgCl_2,1 unit of Taq DNA polymerase and 1×Buffer.Amplification protocol was initial denaturation of 5 min at 95℃;40 cycles of 45 s at 95℃,60 s at 36℃,and 90 s at 72℃and a final cycle of 7 min at 72℃.
2.16 accessions of A.membranaceus germplasms was analyzed by using RAPD markers. 12 polymorphic primers,screened out from 200 random primers,were used and produced 148 reproducible bands of them,127 bands(85.8%) were polymorphic.The cluster analysis divided the germplasm used in this study into 3 groups:wild A.membranaceus,north China A.membranaceus and northeast A.membranaceus.
3.There was significant difference(1%) among different germplasm resources. Polysaccharides,saponins and flavonoids of wild A.membranaceus from Jilin-Helong were significantly higher than cultivated A.membranaceus.In cultivated A.membranaceus, polysaccharides of Jilin-Tonghua was the highest but saponins and flavonoids were lower; saponins and flavonoids of Jilin-Baishan were lower but polysaccharides was the highest. Therefore,in cultivation,we should select different germplasm with different goal.
1.通过模板DNA、引物、dNTP、MgCl_2及Taq DNA聚合酶浓度对RAPD反应探索建立了膜荚黄芪RAPD反应优化体系。即在20μL反应体积中,模板DNA 5ng,引物0.4μmol/L,dNTP 150μmol/L,MgCl_2 2.0 mmol/L,Taq DNA聚合酶0.5 unit,1×Buffer;反应程序为预变性94℃5min,94℃45s、36℃60s、72℃90s共循环40次,最后72℃延伸7min。
2.应用RAPD技术对16份膜荚黄芪种质资源进行分析,从200条随机引物中筛选出12条引物,共扩增出148条带,其中127条为多态带,多态性比率为85.8%,多态性比率较高。对16份膜荚黄芪不同种源进行RAPD扩增,根据遗传距离进行聚类分析结果,将其为3类:第一类为野生黄芪;第二类为华北黄芪,包括山西辉远和内蒙古包头;大部分属于第三类,为东北黄芪。
3.不同种质的有效成分含量差异极显著。野生黄芪中多糖、总皂苷及黄酮含量显著高于所有栽培黄芪;栽培黄芪中,引自吉林通化膜荚黄芪多糖含量最高,但总皂苷和黄酮含量较低;引自吉林白山膜荚黄芪虽然多糖含量较低,但皂苷和黄酮含量在栽培黄芪中最高。因此,需根据不同栽培目的选用不同的种质作为繁殖材料。
RAPD analysis was used to evaluate genetic relationship for 16 accessions germplasm resources of Astragalus membranaceus,collected from abroad and different areas in our country.And effective component analysis was carried out to screen the highest germplsm of effective component for them with UV spectrophotometric method.The aim was to establish the foundation for A.membranaceus breeding and genetic improvement.The main results were summarized as follows.
1.Optimization of RAPD Reaction System for A membranaceus was founded.That was in a 20-ml volume,5 ng template DNA,0.4μmol/L primer,150μmol/L dNTP,2.0 mmol/L MgCl_2,1 unit of Taq DNA polymerase and 1×Buffer.Amplification protocol was initial denaturation of 5 min at 95℃;40 cycles of 45 s at 95℃,60 s at 36℃,and 90 s at 72℃and a final cycle of 7 min at 72℃.
2.16 accessions of A.membranaceus germplasms was analyzed by using RAPD markers. 12 polymorphic primers,screened out from 200 random primers,were used and produced 148 reproducible bands of them,127 bands(85.8%) were polymorphic.The cluster analysis divided the germplasm used in this study into 3 groups:wild A.membranaceus,north China A.membranaceus and northeast A.membranaceus.
3.There was significant difference(1%) among different germplasm resources. Polysaccharides,saponins and flavonoids of wild A.membranaceus from Jilin-Helong were significantly higher than cultivated A.membranaceus.In cultivated A.membranaceus, polysaccharides of Jilin-Tonghua was the highest but saponins and flavonoids were lower; saponins and flavonoids of Jilin-Baishan were lower but polysaccharides was the highest. Therefore,in cultivation,we should select different germplasm with different goal.
引文
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[5]曹福香,田小芳.简述黄芪对免疫疾病的防治作用[J].中华临床医药杂志.2004.5(22):30
[6]孙三省,褚承赤.黄芪药材品种的调查与鉴定[J].中国药学杂志,1990,11:643
[7]严仲恺.中国长白山药用植物彩色图志[M].人民卫生出版社,1997,10:240
[8]肖培根.新编中药志[M].化学工业出版社,2002,876-893
[9]王慧康,叶嘉麟,何侃.内蒙黄芪化学成分研究(Ⅰ)[J].中草药,1987,18(1):5
[10]Chu DT,Wong WL,Mavlight GM.Immunotherapy with Chinese medicinal herbs ⅠImmune restoration of local xenogeneic graft-versus-host reactions in cancer patients by fractionated Astragalus mernbranaceus in vtro[J].J Clin Lab Anal,1988,25:119-123
[11]Zhang X,Xiao HB,Xue XY,et al.Simultaneous characterization of isoflavonoids and astragalosides in two Astragalus species by high-performance liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry[J].J Sep Sci,2007,30(13):2059-2069
[12]Meng L,Qu L,Tang J,et al.A combination of Chinese herbs,Astragalus membranaceus var.mongholicus and Angelica sinensis,enhanced nitric oxide production in obstructed rat kidney[J].Vascul Pharmacol,2007,47(23):174-183
[13]Chu DT,Wong WL,Mavligit GM.Fractionated extract of Astragalus membranaceus,a Chinese medicinal herb,potentiates LAK cell cytotoxicity generated by a low dose of recombinant interleukin-2[J].J Clin Lab Immunol,1988,26:183-187
[14]Chu DT,Wong WL,Mavlight GM.Immunotherapy with Chinese medicinal herbs ⅡReversal cyclophosphamide-induced immune suppression by administration of fractionated Astragalus membranaceus in vivo[J].J Clin Lab Immunol.1988.25125-129
[15]Shen HH,Wang K,Li W,et al.Astragalus Membranaceus prevents airway hyperreactivity in mice related to Th_2 response inhibition[J].J Ethnopharmacol,2008,116(2):363-369
[16]Yu QT,Qi LW,Li P,et al.Determination of seventeen main flavonoids and saponins in the medicinal plant Huang-qi(Radix astragali) by HPLC-DAD-ELSD[J].J Sep Sci,2007,30(9):1292-1299
[17]Cho WC,Leung KN.In vitro and in vivo immunomodulating and immunorestorative effects of Astragalus membranaceus[J].Ethnopharmacol,2007,113(1):132-141
[18]Sun WY,Wei W,Wu L,et al.Effects and mechanisms of extract from Paeonia lactiflora and Astragalus membranaceus on liver fibrosis induced by carbon tetrachloride in rats[J].J Ethnopharmacol,2007,112(3):514-523
[19]Zhu XL,Zhu BD.Mechanisms by which Astragalus membranaceus injection regulates hematopoiesis in myelosuppressed mice[J].Phytother Res,2007,21(7):663-667
[20]黄芪的心血管药理作用[J].中国新药杂志,2003,11:899-901
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