芍药花瓣miRNA提取方法的比较研究
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摘要
小分子RNA(mi RNA)对于调控植物花瓣色泽的形成具有重要作用,而获得高质量的mi RNA是开展相关机理研究的重要前提。本研究采用一步法、多步法及改良多步法对芍药(Paeonia lactiflora)花瓣中mi RNA进行提取,发现一步法和改良多步法均可用于芍药花瓣mi RNA的提取,但相比一步法的价格便宜、提取步骤简单、时间短、提取的RNA浓度较高等优点,改良多步法提取成功率高,提取的芍药花瓣RNA纯度高、完整性好,并且后续的mi RNA前体序列扩增效果好、表达模式检测稳定,是一种高效的芍药花瓣mi RNA提取方法。
Micro RNA(mi RNA) plays an important role in regulating formation of petal color, so obtaining high-quality mi RNA is an important prerequisite for the study of its related mechanism. In this study, one-step, multi-step and improved multi-step methods were used to extract mi RNA from herbaceous peony(Paeonia lactiflora) petals, and the results show that both one-step and improved multi-step methods could be used to extract mi RNA from herbaceous peony petals. Moreover, the advantages of one-step method were low cost, simple extracted step, short extracted time and high RNA concentration. But compared to one-step method, improved multi-step method had higher successive rates of RNA extraction and purity, the better amplification effect of mi RNA precursor sequence and more stable expression pattern. In conclusion, the improved multi-step method is an efficient extraction method for extracting mi RNA from herbaceous peony petals.
Micro RNA(mi RNA) plays an important role in regulating formation of petal color, so obtaining high-quality mi RNA is an important prerequisite for the study of its related mechanism. In this study, one-step, multi-step and improved multi-step methods were used to extract mi RNA from herbaceous peony(Paeonia lactiflora) petals, and the results show that both one-step and improved multi-step methods could be used to extract mi RNA from herbaceous peony petals. Moreover, the advantages of one-step method were low cost, simple extracted step, short extracted time and high RNA concentration. But compared to one-step method, improved multi-step method had higher successive rates of RNA extraction and purity, the better amplification effect of mi RNA precursor sequence and more stable expression pattern. In conclusion, the improved multi-step method is an efficient extraction method for extracting mi RNA from herbaceous peony petals.
引文
Chen CG,Chen QY,Guo JW,Ma ZJ,Liu M,Zhao QY,Zhang YF(2015).Comparing the serum mi RNA extraction effect of three method.Lab Med Clin,12(20):2976-2978(in Chinese with English abstract)[陈昌国,陈秋圆,郭建巍,马志家,刘敏,赵强元,张雅(2015).三种方法提取血清mi RNA效果的比较.检验医学与临床,12(20):2976-2978]
Gou JY,Felippes FF,Liu CJ,Weigel D,Wang JW(2011).Negative regulation of anthocyanin biosynthesis in Arabidopsis by a mi R156-targeted SPL transcription factor.Plant Cell,23(4):1512-1522
Hsieh LC,Lin SI,Shih ACC,Chen JW,Lin WY,Tseng CY,Li WH,Chiou TJ(2009).Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing.Plant Physiol,151(4):2120-2132
Huang FL,Li CH,Sun TT,Gao XQ,Hu BZ(2005).Extraction of total RNA in petals of Paeonia lactiflora Pall.Lett Biotechnol,16(3):282-283(in Chinese with English abstract)[黄凤兰,李长海,孙婷婷,高秀琴,胡宝忠(2005).芍药花瓣总RNA的提取.生物技术通讯,16(3):282-283]
Jia N,Shu QY,Wang LS,Du H,Xu YJ,Liu ZA(2008).Analysis of petal anthocyanins to investigate coloration mechanism in herbaceous peony cultivars.Sci Hortic,117(2):167-173
Jia XY,Yu ZQ,Liang JP,Tang LG,Jin LH,Zhang L,He LH,Li RZ(2013).Cloning of Arabidopsis At-pri-mi R828 gene and its genetic transformation into tomato.Acta Hortic Sin,40(12):2419-2428(in Chinese with English abstract)[贾小云,于治芹,梁建萍,唐贵良,金雷皓,张莉,贺立恒,李润植(2013).拟南芥At-pri-mi R828基因的克隆及其对番茄的遗传转化.园艺学报,40(12):2419-2428]
Li Q,Wang J,Zhao ZX,Shang X(2013).Comparison of three kinds of total RNA extraction method of pansy petals.Jiangsu Agr Sci,41(5):24-26(in Chinese)[李琴,王健,赵钟鑫,尚啸(2013).三色堇花瓣总RNA 3种提取方法的比较.江苏农业科学,41(5):24-26]
LüDJ,Chen J,Zhong Y,Nan P(2014).Compare with two protocols for micro RNA extraction from Astragalus membranaceus var.mongholicus.Plant Physiol J,50(8):1255-1258(in Chinese with English abstract)[吕帝瑾,陈婧,钟扬,南蓬(2014).两种提取蒙古黄芪mi RNA方法的比较.植物生理学报,50(8):1255-1258]
Luo M,Li R,Zhang C,Luo B(2013).Improvement of extraction method of mi RNA from plant tissue with abundant polysaccharides.J Luzhou Med Coll,36(1):6-9(in Chinese with English abstract)[罗茂,李蓉,张春,罗波(2013).富含多糖的植物组织mi RNA提取方法的改进.泸州医学院学报,36(1):6-9]
Vaucheret H(2006).Post-transcriptional small RNA pathways in plants:mechanisms and regulations.Gene Dev,20(7):759-771
Wu T,Lan ZQ(2013).Comparison and analysis of methods of extracting total RNA from petals of Camellia sasanqua.Chin Agr Sci Bull,29(28):129-133(in Chinese with English abstract)[吴田,蓝增全(2013).茶梅花瓣总RNA提取方法的比较和分析.中国农学通报,29(28):129-133]
Xu Q,Liu YL,Zhu AD,Wu XM,Ye JL,Yu KQ,Guo WW,Deng XX(2010).Discovery and comparative profiling of micro RNAs in a sweet orange red-flesh mutant and its wild type.BMC Genomics,11:246
Xu X,He LH(2012).Comparison of current laboratory used of mi R-NA extraction.J North Pharm,9(7):31-32(in Chinese with English abstract)[徐翔,贺丽卉(2012).血清中mi RNA常用提取方法的综合比较.北方药学,9(7):31-32]
Zhang X,Guan LL,Han LL,Yang J,Du LN,Li XK(2015).Study on total RNA extraction from safflower petals.Heilongjiang Agr Sci,2:14-18(in Chinese with English abstract)[张雪,官丽莉,韩怡来,杨晶,杜林娜,李校堃(2015).红花花瓣总RNA提取方法的比较研究.黑龙江农业科学,2:14-18]
Zhao DQ,Jiang Y,Ning CL,Meng JS,Lin SS,Ding W,Tao J(2014).Transcriptome sequencing of a chimaera reveals coordinated expression of anthocyanin biosynthetic genes mediating yellow formation in herbaceous peony(Paeonia lactiflora Pall.).BMCGenomics,15:689
Zhao DQ,Tao J,Han CX,Ge JT(2012).Flower color diversity revealed by differential expression of flavonoid biosynthetic genes and flavonoid accumulation in herbaceous peony(Paeonia lactiflora Pall.).Mol Biol Rep,39(12):11263-11275
Gou JY,Felippes FF,Liu CJ,Weigel D,Wang JW(2011).Negative regulation of anthocyanin biosynthesis in Arabidopsis by a mi R156-targeted SPL transcription factor.Plant Cell,23(4):1512-1522
Hsieh LC,Lin SI,Shih ACC,Chen JW,Lin WY,Tseng CY,Li WH,Chiou TJ(2009).Uncovering small RNA-mediated responses to phosphate deficiency in Arabidopsis by deep sequencing.Plant Physiol,151(4):2120-2132
Huang FL,Li CH,Sun TT,Gao XQ,Hu BZ(2005).Extraction of total RNA in petals of Paeonia lactiflora Pall.Lett Biotechnol,16(3):282-283(in Chinese with English abstract)[黄凤兰,李长海,孙婷婷,高秀琴,胡宝忠(2005).芍药花瓣总RNA的提取.生物技术通讯,16(3):282-283]
Jia N,Shu QY,Wang LS,Du H,Xu YJ,Liu ZA(2008).Analysis of petal anthocyanins to investigate coloration mechanism in herbaceous peony cultivars.Sci Hortic,117(2):167-173
Jia XY,Yu ZQ,Liang JP,Tang LG,Jin LH,Zhang L,He LH,Li RZ(2013).Cloning of Arabidopsis At-pri-mi R828 gene and its genetic transformation into tomato.Acta Hortic Sin,40(12):2419-2428(in Chinese with English abstract)[贾小云,于治芹,梁建萍,唐贵良,金雷皓,张莉,贺立恒,李润植(2013).拟南芥At-pri-mi R828基因的克隆及其对番茄的遗传转化.园艺学报,40(12):2419-2428]
Li Q,Wang J,Zhao ZX,Shang X(2013).Comparison of three kinds of total RNA extraction method of pansy petals.Jiangsu Agr Sci,41(5):24-26(in Chinese)[李琴,王健,赵钟鑫,尚啸(2013).三色堇花瓣总RNA 3种提取方法的比较.江苏农业科学,41(5):24-26]
LüDJ,Chen J,Zhong Y,Nan P(2014).Compare with two protocols for micro RNA extraction from Astragalus membranaceus var.mongholicus.Plant Physiol J,50(8):1255-1258(in Chinese with English abstract)[吕帝瑾,陈婧,钟扬,南蓬(2014).两种提取蒙古黄芪mi RNA方法的比较.植物生理学报,50(8):1255-1258]
Luo M,Li R,Zhang C,Luo B(2013).Improvement of extraction method of mi RNA from plant tissue with abundant polysaccharides.J Luzhou Med Coll,36(1):6-9(in Chinese with English abstract)[罗茂,李蓉,张春,罗波(2013).富含多糖的植物组织mi RNA提取方法的改进.泸州医学院学报,36(1):6-9]
Vaucheret H(2006).Post-transcriptional small RNA pathways in plants:mechanisms and regulations.Gene Dev,20(7):759-771
Wu T,Lan ZQ(2013).Comparison and analysis of methods of extracting total RNA from petals of Camellia sasanqua.Chin Agr Sci Bull,29(28):129-133(in Chinese with English abstract)[吴田,蓝增全(2013).茶梅花瓣总RNA提取方法的比较和分析.中国农学通报,29(28):129-133]
Xu Q,Liu YL,Zhu AD,Wu XM,Ye JL,Yu KQ,Guo WW,Deng XX(2010).Discovery and comparative profiling of micro RNAs in a sweet orange red-flesh mutant and its wild type.BMC Genomics,11:246
Xu X,He LH(2012).Comparison of current laboratory used of mi R-NA extraction.J North Pharm,9(7):31-32(in Chinese with English abstract)[徐翔,贺丽卉(2012).血清中mi RNA常用提取方法的综合比较.北方药学,9(7):31-32]
Zhang X,Guan LL,Han LL,Yang J,Du LN,Li XK(2015).Study on total RNA extraction from safflower petals.Heilongjiang Agr Sci,2:14-18(in Chinese with English abstract)[张雪,官丽莉,韩怡来,杨晶,杜林娜,李校堃(2015).红花花瓣总RNA提取方法的比较研究.黑龙江农业科学,2:14-18]
Zhao DQ,Jiang Y,Ning CL,Meng JS,Lin SS,Ding W,Tao J(2014).Transcriptome sequencing of a chimaera reveals coordinated expression of anthocyanin biosynthetic genes mediating yellow formation in herbaceous peony(Paeonia lactiflora Pall.).BMCGenomics,15:689
Zhao DQ,Tao J,Han CX,Ge JT(2012).Flower color diversity revealed by differential expression of flavonoid biosynthetic genes and flavonoid accumulation in herbaceous peony(Paeonia lactiflora Pall.).Mol Biol Rep,39(12):11263-11275