基于改进的Southern杂交法检测古树木端粒长度的研究
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摘要
研究旨在利用DNA印迹法测量端粒长度从而预测植物的年龄,但木本植物体内富含黄酮类、酚类、多糖、萜类等次生代谢物质,严重影响了提取的基因组DNA产量和质量,以及后续的酶切及杂交实验结果。笔者以北京颐和园5种古树为实验材料,在传统CTAB法提取DNA基础上,不同树种分别添加2%~6%PVP,可有效去除次生代谢物质对DNA的干扰,可被限制性内切酶HinfⅠ酶切完全。同时,对DNA印迹法的其他实验参数如DNA量、曝光时间进行了优化,并进行了3种不同年龄古树端粒长度测定,结果显示古树的平均端粒长度要高于幼树,本研究建立的实验方法为多年生林木特别是古树端粒长度、树龄检测与寿命预测等提供了参考。
Southern hybridization assay is reliable for detecting telomere length to predict plant age. However,secondary metabolites such as flavonoids, phenols, polysaccharides and terpenoids are rich in woody plants,which seriously affect the yield and quality of the extracted genomic DNA as well as subsequent results of enzyme digestion and hybridization. In this research, we modified the method of telomere length measuring by using 5 kinds of ancient trees collected from the Summer Palace in Beijing, on the basis of the conventional CTAB method, 2%-6% PVP(polyvinylpyrrolidone) was added to the DNA extracts of different tree species,which effectively removed the interference of secondary metabolites, therefore DNA could be digested completely by restriction enzyme HinfⅠ. At the same time, we also adjusted DNA content and the exposure time for the Southern hybridization assay, and detected telomere lengths of 3 kinds of trees. The results show that the average telomere length of ancient trees is longer than that of young trees. The improved Southern blotting assay is constructed and lays a foundation for predicting tree age related to telomere length.
Southern hybridization assay is reliable for detecting telomere length to predict plant age. However,secondary metabolites such as flavonoids, phenols, polysaccharides and terpenoids are rich in woody plants,which seriously affect the yield and quality of the extracted genomic DNA as well as subsequent results of enzyme digestion and hybridization. In this research, we modified the method of telomere length measuring by using 5 kinds of ancient trees collected from the Summer Palace in Beijing, on the basis of the conventional CTAB method, 2%-6% PVP(polyvinylpyrrolidone) was added to the DNA extracts of different tree species,which effectively removed the interference of secondary metabolites, therefore DNA could be digested completely by restriction enzyme HinfⅠ. At the same time, we also adjusted DNA content and the exposure time for the Southern hybridization assay, and detected telomere lengths of 3 kinds of trees. The results show that the average telomere length of ancient trees is longer than that of young trees. The improved Southern blotting assay is constructed and lays a foundation for predicting tree age related to telomere length.
引文
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[14]Lapham K,Kvale M N,Lin J,et al.Automated assay of telomere length measurement and informatics for 100,000 subjects in the genetic epidemiology research on adult health and aging(GERA)cohort[J].Genetics,2015,200:1061-1072.
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[16]Alter B P,Rosenberg P S,Giri N,et al.Telomere length is associated with disease severity and declines with age in dyskeratosis congenita[J].Haematologica,2012,97:353-359.
[17]Britt-Compton B,Rowson J,Locke M,et al.Structural stability and chromosome-specific telomere length is governed by cis-acting determinantsinhumans[J].Human Molecular Genetics,2006,15:25-733.
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[19]Flanary B E,Kletetschka G.Analysis of telomere length and telomerase activity in tree species of various life-spans,and with age in the bristlecone pine Pinus longaeva[J].Biogerontology,2005,6:101-111.
[20]Liu D,Qiao N,Song H,et al.Comparative analysis of telomeric restriction fragment lengths in different tissues of Ginkgobiloba trees of different age[J].J Plant Res,2007,120(4):523-528.
[21]李金璐,王硕,于婧,等.一种改良的植物DNA提取方法,植物学报,2013,48(1):72-78.
[22]王关林,方宏筠.植物基因工程原理与技术[M].北京:科学出版社,1998:611-613.
[23]Song H,Liu D,Chen X,et al.Change of season-specific telomere lengths in Ginkgo bilobal[J].Mol Biol Rep,2010,37:819-824.
[24]Lauzon W,Sanchez D J,Cameron D W,et al.Flow cytometric measurement of telomere length[J].Cytometry,2000,42:159-164.
[25]郑广顺.古树快繁及端粒长度与树木年龄关系的研究[D].北京:北京林业大学,2012.
[26]王卓伟,余茂德,鲁成.PVP在桑叶总DNA提取中的应用[J].西南农业大学学报,2001,23(1):61.
[27]胡旭东,撖静宜,要笑云,等.油松端粒长度与树龄相关关系研究[J].广东农业科学,2015,42(12):61-65.
[28]Amani J,Kazemi R,Abbasi A R,et al.A simple and rapid leaf genomic DNA extraction method for polymerase chain reaction analysis[J].Iran J Biotechnol,2011,9(1):69-71.
[29]Rachmayanti Y,Leinemann L,Gailing O,et al.Extraction,amplification and characterization of wood DNA from Dipterocarpaceae[J].Plant Molecular Biology Reporter,2006,24(1):45-55.
[30]Fu R Z,Wang J,Sun Y R,et al.Extraction of genomic DNA suitable for PCR analysis from dried plant rhizomes/roots[J].Biotechnique,1998,25(5):796.
[31]陈昆松,李方,徐昌杰,等.改良CTAB法用于多年生植物组织基因组DNA的大量提取[J].遗传,2004,26(4):529-531.
[32]张徐俞,王瑾瑜,郑广顺,等.四种树木端粒酶活性与树龄及叶位的相关性[J].华北农学报,2014,29(b12):197-201.
[33]Aronen T,Ryyn?nen L.Variation in telomeric repeats of Scots pine(Pinus sylvestris L.)[J].Tree Genetics&Genomes,2012,8(2):267-275.
[34]王钦美,张志宏,崔建国.无性繁殖植株的生理年龄——由端粒长度引发的思考[J].林业科学研究,2017,30(5):866-870.
[35]高凌云,李国栋,童坦君.DNA印迹法和实时定量PCR法在端粒长度测定中的应用比较[J].北京大学学报:医学版,2013,45(2):297-302.
[2]王瑾瑜,张徐俞,王雅群,等.用改进的TRAP法测定树木端粒酶活性[J].应用与环境生物学报,2012,18(4):682-686.
[3]张徐俞,王瑾瑜,郑广顺,等.盐胁迫下沙冬青细胞端粒酶活性的变化与DNA稳定性的关系[J].生物技术通报,2014(10):134-138.
[4]刘颖,吴晓飞,门璟煜,等.端粒酶的非端粒功能研究进展[J].中国细胞生物学学报,2016(5):640-646.
[5]吴晓飞,王瑾瑜,张徐俞,等.盐胁迫下胡杨和合作杨悬浮细胞端粒酶与抵御氧化损伤的关系[J].生物技术通报,2017,33(8):111-119.
[6]Christopher I,Hamer D J,Terry B,et al.Telomere length and age in pinnipeds:The endangered Australian sea lion as a case study[J].Marine Mammal Science,2011,27(4):841-851.
[7]Heaphy C M,Meeker A K.The potential utility of telomererelated markers for cancer diagnosis[J].J Cell Mol Med,2011,15(6):1227-1238.
[8]Dvo?á?kováM,FojtováM,Fajkus J.Chromatin dynamics of plant telomeres and ribosomal genes[J].Plant Journal,2015,83(1):18-37.
[9]Gutierrez C,Puchta H.Chromatin and development:a special issue[J].Plant Journal for Cell&Molecular Biology,2015,83(1):1-3.
[10]Liang J,Jiang C,Peng H,et al.Analysis of the age of Panax ginseng based on telomere length and telomerase activity[J].Scientific Reports,2015,5:7985-7985.
[11]吴斡宁,万涛.浅谈树木年龄测定方法的研究进展[J].绿色科技,2013(7):152-155.
[12]Mender I,Shay J W.Telomere restriction fragment(TRF)analysis[J].Bio-Protocol,2015,5(22):e1658.
[13]张彬,王长利.端粒长度检测方法及其应用[J].生物技术通报,2016,32(11):93-98.
[14]Lapham K,Kvale M N,Lin J,et al.Automated assay of telomere length measurement and informatics for 100,000 subjects in the genetic epidemiology research on adult health and aging(GERA)cohort[J].Genetics,2015,200:1061-1072.
[15]Toutain J,Prochazkova-Carlotti M,Horovitz J,et al.Evaluation of Quantitative Fluorescence in situ hybridization for relative measurement of telomere length in placental mesenchymal core cells[J].Gynecologic and Obstetric Investigation,2016,81:54-60.
[16]Alter B P,Rosenberg P S,Giri N,et al.Telomere length is associated with disease severity and declines with age in dyskeratosis congenita[J].Haematologica,2012,97:353-359.
[17]Britt-Compton B,Rowson J,Locke M,et al.Structural stability and chromosome-specific telomere length is governed by cis-acting determinantsinhumans[J].Human Molecular Genetics,2006,15:25-733.
[18]Martin-Ruiz C M,Baird D,Roger L,et al.Reproducibility of telomere length assessment:an international collaborative study[J].International Journal of Epidemiology,2015,44:1673-1683.
[19]Flanary B E,Kletetschka G.Analysis of telomere length and telomerase activity in tree species of various life-spans,and with age in the bristlecone pine Pinus longaeva[J].Biogerontology,2005,6:101-111.
[20]Liu D,Qiao N,Song H,et al.Comparative analysis of telomeric restriction fragment lengths in different tissues of Ginkgobiloba trees of different age[J].J Plant Res,2007,120(4):523-528.
[21]李金璐,王硕,于婧,等.一种改良的植物DNA提取方法,植物学报,2013,48(1):72-78.
[22]王关林,方宏筠.植物基因工程原理与技术[M].北京:科学出版社,1998:611-613.
[23]Song H,Liu D,Chen X,et al.Change of season-specific telomere lengths in Ginkgo bilobal[J].Mol Biol Rep,2010,37:819-824.
[24]Lauzon W,Sanchez D J,Cameron D W,et al.Flow cytometric measurement of telomere length[J].Cytometry,2000,42:159-164.
[25]郑广顺.古树快繁及端粒长度与树木年龄关系的研究[D].北京:北京林业大学,2012.
[26]王卓伟,余茂德,鲁成.PVP在桑叶总DNA提取中的应用[J].西南农业大学学报,2001,23(1):61.
[27]胡旭东,撖静宜,要笑云,等.油松端粒长度与树龄相关关系研究[J].广东农业科学,2015,42(12):61-65.
[28]Amani J,Kazemi R,Abbasi A R,et al.A simple and rapid leaf genomic DNA extraction method for polymerase chain reaction analysis[J].Iran J Biotechnol,2011,9(1):69-71.
[29]Rachmayanti Y,Leinemann L,Gailing O,et al.Extraction,amplification and characterization of wood DNA from Dipterocarpaceae[J].Plant Molecular Biology Reporter,2006,24(1):45-55.
[30]Fu R Z,Wang J,Sun Y R,et al.Extraction of genomic DNA suitable for PCR analysis from dried plant rhizomes/roots[J].Biotechnique,1998,25(5):796.
[31]陈昆松,李方,徐昌杰,等.改良CTAB法用于多年生植物组织基因组DNA的大量提取[J].遗传,2004,26(4):529-531.
[32]张徐俞,王瑾瑜,郑广顺,等.四种树木端粒酶活性与树龄及叶位的相关性[J].华北农学报,2014,29(b12):197-201.
[33]Aronen T,Ryyn?nen L.Variation in telomeric repeats of Scots pine(Pinus sylvestris L.)[J].Tree Genetics&Genomes,2012,8(2):267-275.
[34]王钦美,张志宏,崔建国.无性繁殖植株的生理年龄——由端粒长度引发的思考[J].林业科学研究,2017,30(5):866-870.
[35]高凌云,李国栋,童坦君.DNA印迹法和实时定量PCR法在端粒长度测定中的应用比较[J].北京大学学报:医学版,2013,45(2):297-302.