药用植物苦参EST-SSR标记的开发及DNA指纹图谱的构建
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摘要
为挖掘苦参优异种质资源,利用大宗药材苦参现有的EST资源开发EST-SSR标记,分析不同地理种源苦参的遗传多样性,构建其特有的DNA指纹图谱。通过生物信息学手段对NCBI数据库中苦参EST序列进行SSR查找,利用Primer 5.0软件设计EST-SSR引物,筛选多态性引物检测苦参样本DNA差异,采用UPGMA聚类分析样本的遗传多样性,并构建DNA指纹图谱。经分析得到92条Unigene,从中发掘出126个EST-SSR位点分布于61条Unigene上。SSR检出率为66.30%,平均分布距离为0.512kb;共搜索到61种EST-SSR的重复基元,五核苷酸和六核苷酸重复是主要的重复类型,二者出现频率分别为68.25%和25.40%,没有三核苷酸出现,AAAAT/ATTTT与AAGTGG/CCACTT是五、六核苷酸的优势重复类型;设计出65对苦参的EST-SSR引物,随机合成26对,筛选出18对多态性引物可用于苦参样本遗传多样性分析,共检测出77个等位变异,平均每对引物检测出4.3个基因位点,引物多态性比率为69.2%;聚类分析结果表明:苦参材料首先按照居群聚类,在相似系数为0.73处,供试材料按亲缘关系远近分为5大类,相同或相近产地来源的苦参样本聚为一类,呈现出一定的地域性分布规律;筛选到2对核心引物DYH011与DYH018,构建了苦参样本的DNA指纹图谱,作为对不同产地来源苦参鉴定和溯源的依据。
In order to explore the elite germplasm resources of Sophora flavescen,EST-SSR markers were developed using the existing EST resources,the genetic diversity of the herb from various geographic locations was analyzed,and their DNA fingerprints were constructed.SSR information was obtained from NCBI by bioinformatics methods,and ESTSSR primers were designed by the software Primer 5.0 Plus.The DNA samples of Sophora flavescens were used as templates in PCR amplification with polymorphic primers.Their genetic diversity was analyzed based on UPGMA clustering.DNA fingerprints were constructed.A total of 92 Unigenes were obtained from Sophora flavescentis EST data bank,among which 126 simple sequence repeats were distributed in 61 Unigenes accounting for 66.30% of thetotal number of Unigenes.The frequency of SSRs was 0.512 kb.Penta-nucleotide and hexa-nucleotide repeats were the dominant types among the obtained Unigenes accounting for 68.05% and 25.40%,respectively.No trinucleotide appeared.AAAAT/ATTTT and AAGTGG/CCACTT were respectively the most abundant motifs for penta-nucleotide and hexa-nucleotide motifs.The 65 pair primers were designed by Primer 5.0 and 26 pairs of which were randomly synthesized.Among them,18 pairs produced clear and stable bands.The genetic diversity of 48 samples from 12 geographical populations was investigated.A total 77 loci with an average of 4.3 loci for each pair of primers were detected,and the polymorphic primer ratio was 69.2%.The materials were divided into 5 groups according to the genetic relationship in Clustering.Materials with the same or close origins were clustered into one group,indicating their geographical distribution characteristics.Two pairs of core primers DYH011 and DYH018 were selected,and the DNA fingerprints of Sophora flavescens were constructed,which could be used as a basis for the rapid and accurate identification and traceability of different geographical sources.
In order to explore the elite germplasm resources of Sophora flavescen,EST-SSR markers were developed using the existing EST resources,the genetic diversity of the herb from various geographic locations was analyzed,and their DNA fingerprints were constructed.SSR information was obtained from NCBI by bioinformatics methods,and ESTSSR primers were designed by the software Primer 5.0 Plus.The DNA samples of Sophora flavescens were used as templates in PCR amplification with polymorphic primers.Their genetic diversity was analyzed based on UPGMA clustering.DNA fingerprints were constructed.A total of 92 Unigenes were obtained from Sophora flavescentis EST data bank,among which 126 simple sequence repeats were distributed in 61 Unigenes accounting for 66.30% of thetotal number of Unigenes.The frequency of SSRs was 0.512 kb.Penta-nucleotide and hexa-nucleotide repeats were the dominant types among the obtained Unigenes accounting for 68.05% and 25.40%,respectively.No trinucleotide appeared.AAAAT/ATTTT and AAGTGG/CCACTT were respectively the most abundant motifs for penta-nucleotide and hexa-nucleotide motifs.The 65 pair primers were designed by Primer 5.0 and 26 pairs of which were randomly synthesized.Among them,18 pairs produced clear and stable bands.The genetic diversity of 48 samples from 12 geographical populations was investigated.A total 77 loci with an average of 4.3 loci for each pair of primers were detected,and the polymorphic primer ratio was 69.2%.The materials were divided into 5 groups according to the genetic relationship in Clustering.Materials with the same or close origins were clustered into one group,indicating their geographical distribution characteristics.Two pairs of core primers DYH011 and DYH018 were selected,and the DNA fingerprints of Sophora flavescens were constructed,which could be used as a basis for the rapid and accurate identification and traceability of different geographical sources.
引文
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[4]刘小艳,方红,羊正纲,滕理送,王懿娜,丁颖果,蒋筱凌.苦参碱对人恶性黑色瘤A375细胞株増值和凋亡的影响[J].中国药学杂志,2007,42(18):1377-1379Liu X Y,Fang H,Yang Z G,Teng L S,Wang Y N,Ding YG,Jiang X L.Effects of matrine on the proliferation and apoptosis induction in human malignant melanoma cell Line A375[J].Chinese Pharmaceutical Journal,2007,42(18):1377-1379(in Chinese)
[5]任广慧,黄伟,肖旭,石文达,郑芳芳,高岭.氧化苦参碱对胶原蛋白诱导大鼠关节炎症及T细胞亚群的影响[J].中国药学杂志,2012,47(5):350-353Ren G H,Huang W,Xiao X,Shi W D,Zheng F F,Gao L.Effects of oxymatrine on joint inflammation and T cell subsets in rats with collagen induced arthritis[J].Chinese Pharmaceutical Journal,2012,47:350-353(in Chinese)
[6]战渤玉,李东霞,高明.苦参的现代研究进展[J].中医药信息,2009,26(1):23-25Zhan B Y,Li D X,Gao M.Modern research progress of Sophora flavescens Ait[J].Information on Traditional Chinese Medieine,2009,26(1):23-25(in Chinese)
[7]邹林有,陈垣.不同处理方法对苦参种子发芽特性的影响[J].甘肃农业大学学报,2008,43(5):80-83Zou L Y,Chen Y.Effect of different treatment methods on seed germination characteristics of Sophoraflavescens[J].Journal of Gansu Agricultural University,2008,43(5):80-83(in Chinese)
[8]吴尚英,李安平,关扎根,魏红国.不同种源苦参种子生物学特性的研究[J].种子,2011,31(11):70-72Wu S Y,Li A P,Guan Z G,Wei H G.A Study on biological characterstics of Sophora flavescens Ait seeds from different provenances[J].Seed,2012,31(11):70-72(in Chinese)
[9]陈静,王淑美,孟江,孙飞,王佰灵,刘贝,梁生旺.不同生长年限苦参不同部位的生物碱含量[J].中国实验方剂学杂志,2013,19(7):80-84Chen J,Wang S M,Meng J,Sun F,Wang B L,Liu B,Liang S W.Determination of five alkaloids fromSophora flavescens of different parts and different growth years[J].Chinese Journal of Experimental Traditional Medical Formulae,2013,19(7):80-84(in Chinese)
[10]李威,梁宏,尹婷,王邠,赵玉英.中药苦参主要黄酮类成分的研究[J].药学学报,2008,43(28):833-837Li W,Liang H,Yin T,Wang B,Zhao Y Y.Main flavonoids from Sophora flavescenes[J].Acta Pharmaceutica Sinica,2008,43(28):833-837(in Chinese)
[11]于海红,姜音,丛景香,吴秀红.苦参粗提液中苦参碱和氧化苦参碱的高效液相色谱法测定[J].时珍国医国药,2013,24(1):126-127Yu H H,Jiang Y,Cong J X,Wu X H.Determination of matrine and oxymatrine in the crude extract of Sophora flavescens by HPLC[J].Lishizhen Medicine and Materia Medica Research,2013,24(1):126-127(in Chinese)
[12]张蕾,王志伟,廉建伟,周浩,陈晓辉,毕开顺.HPLC-MS法同时测定大鼠血浆中苦参碱、氧化苦参碱和氧化槐果碱的浓度及其药代动力学[J].药学学报,2008,43(8):843-847Zhang L,Wang Z W,Lian J W,Zhou H,Chen X H,Bi KS.Simultaneous determination of matrine,oxysophocarpin and oxymatrine in rat plasma by HPLC-MS and its application in the pharmacokinetic study[J].Acta Pharmaceutica Sinica,2008,43(8):843-847(in Chinese)
[13]周延清.DNA分子标记技术在植物研究中的应用[M].北京:化学工业出版社,2005,186-187Zhou Y Q.Application of DNA Molecular Markers Technology In Plant Research[M].Beijing:Chemical Industry Press,2005,186-187(in Chinese)
[14]Liu Y L,Li Y H,Zhou G A,Uzokwe N,Chang R Z,Chen S Y,Qiu L J.Development of Soybean EST-SSR markers and their use to assess genetic diversity in the Subgenus Soja[J].Agricultural Sciences in China,2010,9(10):1423-1429
[15]Xu W,Yang Q,Huai H Y,Liu A Z.Development of EST-SSR markers and investigation of genetic relatedness in tung tree[J].Tree Genetics&Genomes,2012,8(4):933-940
[16]Kayesh E.Zhang Y Y,Liu G S,Bilkish N,Sun X,Leng XP.Development of highly polymorphic EST-SSR markers and segregation in F1hybrid population of Vitis vinifera L[J].Genetics and Molecular Research,2013,12(3):3871-3878
[17]潘海涛,汪俊君,王盈盈,齐照良,李斯深.小麦EST-SSR标记的开发和遗传作图[J].中国农业科学,2010,43(3):452-461Pan H T,Wang J J,Wang Y Y,Qi Z L,Li S S.Development and mapping of EST-SSR markers in wheat[J].Scientia Agricultura Sinica,2010,43(3):452-461(in Chinese)
[18]王月,刘晓庆,李昊,董学会.EST-SSR标记应用于麦冬类植物遗传多样性研究[J].中国农业大学学报,2017,22(9):12-20Wang Y,Liu X Q,Li H,Dong X H.Genetic diversity of Liriopogons revealed by EST-SSR Markers[J].Journal of China Agricultural University,2017,22(9):12-20(in Chinese)
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