杜梨不同组织总RNA提取方法比较及质量分析
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摘要
【目的】针对杜梨组织中富含多酚、多糖等次生代谢物质特点,筛选从杜梨不同组织中提取高质量RNA的方法。【方法】以杜梨不同组织(根、茎、叶、花和果)为材料,分别采用Trizol法、改良CTAB法和RNAprep Pure Plant Kit试剂盒法提取总RNA,利用琼脂糖凝胶电泳和NanoDrop 2000微量分光光度计对RNA进行质量检测。【结果】3种方法均能提出杜梨不同组织中总RNA,但RNAprep Pure Plant Kit法提取的总RNA电泳条带清晰、完整性较好,根、茎、叶、花和果提取的RNA纯度分别为2.07、2.02、2.01、2.01和2.02;浓度分别为186.2 ng·μL~(-1)、174.5 ng·μL~(-1)、163.6 ng·μL~(-1)、167.3 ng·μL~(-1)和121.1 ng·μL~(-1)。以RNAprep Pure Plant Kit试剂盒法提取的总RNA反转录成cDNA为模板,内参基因PbUBI为目的基因进行RT-PCR扩增,均可获得明亮单一条带。【结论】RNAprep Pure Plant Kit试剂盒法更适用于杜梨不同组织总RNA提取,可为下一步进行基因克隆及功能验证等分子生物学研究奠定基础。
【Objective】Aiming at the abundant amounts in amylose,hydroxybenzene and other secondary metabolities in the tissues of Pyrus betulaefolia Bunge,the study was undertaken to screen out the method in order to obtain the high-quality RNA.【Methods】The different tissues(root,stem,leaf,flower and fruit) were used as the materials,and the total RNA was extracted by Trizol method,the improved CTAB method and RNAprep Pure Plant Kit method.The concentration and purity of the extracted RNA were analyzed by Agarose Gel Electrophoresis and NanoDrop 2000.【Results】The total RNA can be extracted by the 3 methods,and the bands of the total RNA extracted by RNAprep Pure Plant Kit method were clear.The purities were 2.07,2.02,2.01,2.01 and 2.02,respectively,and the concentrations were186.2 ng· μ L~(-1),174.5 ng· μ L~(-1),163.6 ng· μ L~(-1),167.3 ng· μ L~(-1) and 121.1 ng· μ L~(-1) respectively.Additionally,the total RNA extracted with RNAprep Pure Plant Kit method could be used for reverse transcription,and the amplification bands of PbUBI gene were specific.【Conclusion】The RNAprep Pure Plant Kit method was suitable for different tissues of Pyrus betulaefolia Bunge,which laid the foundation for the further molecular biology research.
【Objective】Aiming at the abundant amounts in amylose,hydroxybenzene and other secondary metabolities in the tissues of Pyrus betulaefolia Bunge,the study was undertaken to screen out the method in order to obtain the high-quality RNA.【Methods】The different tissues(root,stem,leaf,flower and fruit) were used as the materials,and the total RNA was extracted by Trizol method,the improved CTAB method and RNAprep Pure Plant Kit method.The concentration and purity of the extracted RNA were analyzed by Agarose Gel Electrophoresis and NanoDrop 2000.【Results】The total RNA can be extracted by the 3 methods,and the bands of the total RNA extracted by RNAprep Pure Plant Kit method were clear.The purities were 2.07,2.02,2.01,2.01 and 2.02,respectively,and the concentrations were186.2 ng· μ L~(-1),174.5 ng· μ L~(-1),163.6 ng· μ L~(-1),167.3 ng· μ L~(-1) and 121.1 ng· μ L~(-1) respectively.Additionally,the total RNA extracted with RNAprep Pure Plant Kit method could be used for reverse transcription,and the amplification bands of PbUBI gene were specific.【Conclusion】The RNAprep Pure Plant Kit method was suitable for different tissues of Pyrus betulaefolia Bunge,which laid the foundation for the further molecular biology research.
引文
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[14]关玲,赵密珍,王庆莲,吴伟民,钱亚明,巫建华.改良CTAB方法提取果树不同组织的RNA[J].江苏农业科学,2018,46(15):19-22.GUAN Ling,ZHAO Mizhen,WANG Qinglian,WU Weimin,QIAN Yaming,WU Jianhua.Improved CTAB method for extracting RNA from different tissues of fruit trees[J].Jiangsu Agricultural Sciences,2018,46(15):19-22.
[15]尚瑞广,李丽,孙涛,王兵益.玛咖贮藏根总RNA提取方法的比较[J].分子植物育种,2017,15(2):1936-1940.SHANG Ruiguang,LI Li,SUN Tao,WANG Bingyi.Comparison of methods for total RNA extraction from Lepidium meyenii(Maca)storage roots[J].Molecular Plant Breeding,2017,15(2):1936-1940.
[16]孙长悦.梨组织RNA提取方法研究[J].北方园艺,2009(3):19-21.SUN Changyue.The study for extraction methods of different tissues in pear[J].Northern Horticulture,2009(3):19-21.
[17]巩艳明,曹后男,宗成文,金灿,许雪.三种方法提取不同品种梨叶片总RNA[J].湖北农业科学,2011,50(15):3204-3206.GONG Yanming,CAO Hounan,ZONG Chengwen,JIN Can,XU Xue.Three methods for extracting total RNA from leaves of pear varieties[J].Hubei Agricultural Sciences,2011,50(15):3204-3206.
[18]王西成,乔玉山,欧春青,王斐,马力,姜淑苓.三种梨果皮总RNA提取方法的比较研究[J].甘肃农业大学学报,2010,45(2):91-94.WANG Xicheng,QIAO Yushan,OU Chunqing,WANG Fei,MA Li,JIANG Shuling.Extraction of total RNA from pericarp of pears with three methods[J].Journal of Gansu Agricultural University,2010,45(2):91-94.
[19]徐义流,张绍铃.梨花柱RNA提取方法[J].农业生物技术学报,2004,12(6):737-738.XU Yiliu,ZHANG Shaoling.A mehtod for RNA isolation from pear style[J].Journal of Agricultural Biotechnology,2004,12(6):737-738.
[20]XI L,MU T,SUN H.Preparative purification of polyphenols from sweet potato(Ipomoea batatas L.)leaves by AB-8 macroporous resins[J].Food Chemistry,2015,172(172):166-174.
[21]FANG G,HAMMAR S,GRUMET R.A quick and inexpensive method for removing polysaccharides from plant genomic DNA[J].Biotechniques,1992,13(1):52-56.
[22]周敏,付金娥,韦树根,潘丽梅.青蒿不同部位总RNA提取方法比较[J].江苏农业科学,2017,45(3):31-33.ZHOU Min,FU Jine,WEI Shugen,PAN Limei.Comparison of total RNA extraction methods from different parts of Artemisia annua[J].Jiangsu Agricultural Sciences,2017,45(3):31-33.
[23]许让伟.砂梨果实和叶片中糖积累及代谢相关酶活性变化研究[D].武汉:华中农业大学,2009.XU Rangwei.Studies on changes of sugar accumulation and related enzymes activities in sand pear fruits and leaves[D].Wuhan:Huazhong Agricultural University,2009.
[24]李静,聂继云,曹玉芬,李志霞,闫震,毋永龙.砀山酥梨和秋白梨酚类物质UPLC-PDA-MS/MS-ESI分析[J].园艺学报,2016,43(4):752-762.LI Jing,NIE Jiyun,CAO Yufen,LI Zhixia,YAN Zhen,WUYong long.UPLC-PDA-MS/MS-ESI analysis of phenolic compounds in fruits of Dangshan Suli and Qiubaili pears(Pyrus bretschneideri)[J].Acta Horticulturae Sinica,2016,43(4):752-762.
[2]ROBBANI M,BANNO K,KAKEGAWA M.Differential flooding tolerance of some dwarfing pear rootstock clones selected from the progenies of Pyrus betulaefolia and P.calleryana[J].Engei Gakkai Zasshi,2006,75(4):297-305.
[3]宗宇,孙萍,牛庆丰,滕元文.中国北方野生杜梨分布现状及其形态多样性评价[J].果树学报,2013,30(6):918-923.ZONG Yu,SUN Ping,NIU Qingfeng,TENG Yuanwen.Distribution situation and assessment of morphological diversity of wild Pyrus betulaefolia in Northern China[J].Journal of Fruit Science,2013,30(6):918-923.
[4]J.萨姆布鲁克,D.拉塞尔.分子克隆实验指南[M].第三版.北京:科学出版社,2002:516-522.SAMBROOK J,RUSSELL D.Molecular clone:a laboarty manual[M].3rd ed.Beijing:Science Press,2002:516-522.
[5]顾红雅.植物基因与分子操作[M].北京:北京大学出版社,1995:101-115.GU Hongya.Plant genes and molecular manipulations[M].Beijing:Beijing University Press,1995:101-115.
[6]卢圣栋.现代分子生物学实验技术[M].北京:中国协和医科大学出版社,1999:61-66.LU Shengdong.Current protocols for molecular biology[M].Beijing:Peking Union Medical College Press,1999:61-66.
[7]董祯.梨果实ACC氧化酶基因克隆的研究[D].保定:河北农业大学,2007.DONG Zhen.The Study on the the ACC oxidase genes clone of pear fruit[D].Baoding:Hebei Agricultural University,2007.
[8]徐昌杰,陈昆松,张波,王钱洁,叶薇佳.柑橘组织RNA提取方法研究[J].果树学报,2004,21(2):136-140.XU Changjie,CHEN Kunsong,ZHANG Bo,WANG Qianjie,YE Weijia.A study on methods for RNA extraction from citrus tissues[J].Journal of Fruit Science,2004,21(2):136-140.
[9]孙雯,石海燕,张玉星.黄金梨果实高质量总RNA提取方法的研究[J].华北农学报,2013,28(2):70-72.SUN Wen,SHI Haiyan,ZHANG Yuxing.Extraction of high quality RNA from Whangkeumbae pear[J].Acta Agriculturae Boreali-Sinica,2013,28(2):70-72.
[10]赵小亮,白红进,汪河滨,周忠波,罗锋.杜梨果实多糖提取方法及含量测定的研究[J].西北农业学报,2007,16(4):279-281.ZHAO Xiaoliang,BAI Hongjin,WANG Hebin,ZHOU Zhongbo,LUO Feng.Study on the extraction methods and content measurement of polysaccharide from Pyrus betulaefolia Bunge fruit[J].Acta Agriculturae Boreali-occidentalis Sinica,2007,16(4):279-281.
[11]孙艳琼,段红平.植物基因克隆方法在作物上的应用[J].南方农业学报,2016,36(3):275-279.SUN Yanqiong,DUAN Hongping.Application of methods of plant gene cloning in crop[J].Guangxi Agricultural Science,2016,36(3):275-279.
[12]WEBER A P.Discovering new biology through sequencing of RNA[J].Plant Physiology,2015,169(3):1524-1531.
[13]安振宇,何新华,董龙,张树伟,胡颖,罗聪,黄桂香.柠檬总RNA提取方法研究[J].基因组学与应用生物学,2015,34(1):203-207.AN Zhenyu,HE Xinhua,DONG Long,ZHANG Shuwei,HUYing,LUO Cong,HUANG Guixiang.Studies on extraction methods of total RNA from lemon tissues[J].Genomics and Applied Biology,2015,34(1):203-207.
[14]关玲,赵密珍,王庆莲,吴伟民,钱亚明,巫建华.改良CTAB方法提取果树不同组织的RNA[J].江苏农业科学,2018,46(15):19-22.GUAN Ling,ZHAO Mizhen,WANG Qinglian,WU Weimin,QIAN Yaming,WU Jianhua.Improved CTAB method for extracting RNA from different tissues of fruit trees[J].Jiangsu Agricultural Sciences,2018,46(15):19-22.
[15]尚瑞广,李丽,孙涛,王兵益.玛咖贮藏根总RNA提取方法的比较[J].分子植物育种,2017,15(2):1936-1940.SHANG Ruiguang,LI Li,SUN Tao,WANG Bingyi.Comparison of methods for total RNA extraction from Lepidium meyenii(Maca)storage roots[J].Molecular Plant Breeding,2017,15(2):1936-1940.
[16]孙长悦.梨组织RNA提取方法研究[J].北方园艺,2009(3):19-21.SUN Changyue.The study for extraction methods of different tissues in pear[J].Northern Horticulture,2009(3):19-21.
[17]巩艳明,曹后男,宗成文,金灿,许雪.三种方法提取不同品种梨叶片总RNA[J].湖北农业科学,2011,50(15):3204-3206.GONG Yanming,CAO Hounan,ZONG Chengwen,JIN Can,XU Xue.Three methods for extracting total RNA from leaves of pear varieties[J].Hubei Agricultural Sciences,2011,50(15):3204-3206.
[18]王西成,乔玉山,欧春青,王斐,马力,姜淑苓.三种梨果皮总RNA提取方法的比较研究[J].甘肃农业大学学报,2010,45(2):91-94.WANG Xicheng,QIAO Yushan,OU Chunqing,WANG Fei,MA Li,JIANG Shuling.Extraction of total RNA from pericarp of pears with three methods[J].Journal of Gansu Agricultural University,2010,45(2):91-94.
[19]徐义流,张绍铃.梨花柱RNA提取方法[J].农业生物技术学报,2004,12(6):737-738.XU Yiliu,ZHANG Shaoling.A mehtod for RNA isolation from pear style[J].Journal of Agricultural Biotechnology,2004,12(6):737-738.
[20]XI L,MU T,SUN H.Preparative purification of polyphenols from sweet potato(Ipomoea batatas L.)leaves by AB-8 macroporous resins[J].Food Chemistry,2015,172(172):166-174.
[21]FANG G,HAMMAR S,GRUMET R.A quick and inexpensive method for removing polysaccharides from plant genomic DNA[J].Biotechniques,1992,13(1):52-56.
[22]周敏,付金娥,韦树根,潘丽梅.青蒿不同部位总RNA提取方法比较[J].江苏农业科学,2017,45(3):31-33.ZHOU Min,FU Jine,WEI Shugen,PAN Limei.Comparison of total RNA extraction methods from different parts of Artemisia annua[J].Jiangsu Agricultural Sciences,2017,45(3):31-33.
[23]许让伟.砂梨果实和叶片中糖积累及代谢相关酶活性变化研究[D].武汉:华中农业大学,2009.XU Rangwei.Studies on changes of sugar accumulation and related enzymes activities in sand pear fruits and leaves[D].Wuhan:Huazhong Agricultural University,2009.
[24]李静,聂继云,曹玉芬,李志霞,闫震,毋永龙.砀山酥梨和秋白梨酚类物质UPLC-PDA-MS/MS-ESI分析[J].园艺学报,2016,43(4):752-762.LI Jing,NIE Jiyun,CAO Yufen,LI Zhixia,YAN Zhen,WUYong long.UPLC-PDA-MS/MS-ESI analysis of phenolic compounds in fruits of Dangshan Suli and Qiubaili pears(Pyrus bretschneideri)[J].Acta Horticulturae Sinica,2016,43(4):752-762.