一种改良的红豆杉成熟针叶总RNA提取方法
|
摘要
红豆杉(Taxus chinensis(Pilger)Rehd.)是一种重要的药源植物,根据红豆杉针叶特点,本研究提出改良RNA提取方法,即十六烷基三甲基溴化铵-氯化锂(CTAB-Li Cl)联用法,以红豆杉的幼嫩、成熟针叶为材料,采用TRIzol法提取RNA,结果表明,TRIzol法提取嫩针叶中的RNA质量完整,成熟针叶中的RNA质量不完整;针对成熟针叶采用CTAB法、SDS-酚法和改良的CTAB-Li Cl法提取RNA,并进行了质量验证比对显示,CTAB法和SDS-酚法提取的RNA质量差,并存在不同程度的多糖、多酚和DNA污染,改良CTAB-Li Cl联用法提取红豆杉成熟针叶的RNA质量完整,A_(260)/A_(280)比值在1.9~2.0之间,RNA产率最高,分别为CTAB法和SDS-酚法提取的RNA产率的1.37和1.24倍。进一步反转录后,利用特异性引物进行扩增验证,发现扩增的片段条带清晰,片段大小符合预期。成熟针叶中高质量的RNA更有利于红豆杉分子生物学方面的深入研究。
Taxus chinensis( Pilger) Rehd. is one of the most important medicinal plants. According to the characteristics of the needles,a new RNA extraction method,CTAB-Li Cl,was developed. RNA was extracted from young and mature needles of Taxus Chinensis by TRIzol method. The result showed that the quality of RNA extracted from young needles was complete,and that of RNA from mature needles was incomplete. As a comparison,cetyl trimethyl ammonium bromide( CTAB) method,SDS-phenol method and improved CTABLi Cl method were respectively used for extraction of RNA from mature needles. The comparative result showed that the quality of RNA extracted by CTAB method and SDS-phenol method was incomplete,accompanied with different degrees of contamination of polysaccharides,polyphenols and DNA. The improved CTAB-Li Cl method could better extract RNA from mature needles. The ratio of A_(260)/A_(280) was in the range of 1.9-2.0,and the yield of RNA was the highest,which was 1.37 and 1.24 times compared with that by CTAB and SDS phenol extraction,respectively. The reverse transcription c DNA was further tested through PCR with the specific primers. The amplified fragments displayed in clear and bright bands in accordance with the expected size.High quality RNA from mature needles has more advantages in molecular biology study of Taxus chinensis.
Taxus chinensis( Pilger) Rehd. is one of the most important medicinal plants. According to the characteristics of the needles,a new RNA extraction method,CTAB-Li Cl,was developed. RNA was extracted from young and mature needles of Taxus Chinensis by TRIzol method. The result showed that the quality of RNA extracted from young needles was complete,and that of RNA from mature needles was incomplete. As a comparison,cetyl trimethyl ammonium bromide( CTAB) method,SDS-phenol method and improved CTABLi Cl method were respectively used for extraction of RNA from mature needles. The comparative result showed that the quality of RNA extracted by CTAB method and SDS-phenol method was incomplete,accompanied with different degrees of contamination of polysaccharides,polyphenols and DNA. The improved CTAB-Li Cl method could better extract RNA from mature needles. The ratio of A_(260)/A_(280) was in the range of 1.9-2.0,and the yield of RNA was the highest,which was 1.37 and 1.24 times compared with that by CTAB and SDS phenol extraction,respectively. The reverse transcription c DNA was further tested through PCR with the specific primers. The amplified fragments displayed in clear and bright bands in accordance with the expected size.High quality RNA from mature needles has more advantages in molecular biology study of Taxus chinensis.
引文
1 HAN Fei,KANG Lin-Zhi,GUO Li-Qiong,CHEN Xiao-Yang,LIN Jun-Fang.Journal of Food Science and Biotechnology,2011,30(5):728-733韩飞,康林芝,郭丽琼,陈晓阳,林俊芳.食品与生物技术学报,2011,30(5):728-733
2 ZHOU Hua,ZHU Qi,YANG Yan-Fang,YU Fa-Xin,QIU De-You.Plant Physiology Journal,2014,50(4):373-381周华,朱祺,杨艳芳,余发新,邱德有.植物生理学报,2014,50(4):373-381
3 GAO Ming-Bo,ZHANG Wei,LI Xing-Tai,RUAN Cheng-Jiang,FAN Sheng-Di.Chinese Journal of Biotechnology,2011,27(1):101-107高明波,张卫,李兴泰,阮成江,范圣第.生物工程学报,2011,27(1):101-107
4 LI Nai-Wei,SHU Xiao-Chun,HE Shu-Lan,JIANG Yu-Mei,XIA Bing,PENG Feng.Acta Botanica Boreali-Occidentalia Sinica,2010,30(12):2536-2541李乃伟,束晓春,何树兰,江玉梅,夏冰,彭峰.西北植物学报,2010,30(12):2536-2541
5 XIAO Lu,LIU Xian-Hu,LIU Ji-Sheng,LI Mei-Shan,XU Ming-Zi.Scientia Silvae Sinicae,2011,47(5):168-170肖璐,刘宪虎,刘继生,李美善,许明子.林业科学,2011,47(5):168-170
6 WU Qiong,DUAN Xiao-Qun,CHEN Xu,XIAO Pei-Gen.China Journal of Chinese Materia Medica,2012,37(24):3728-3733吴琼,段小群,陈旭,肖培根.中国中药杂志,2012,37(24):3728-3733
7 LI Yan-Lin,YANG Xing-Xing,ZHANG Jia-Yin,HUANG San-Wen,XIONG Xing-Yao.Acta Horticulturae Sinica,2014,41(4):735-745李炎林,杨星星,张家银,黄三文,熊兴耀.园艺学报,2014,41(4):735-745
8 WANG Yu-Cheng.,YANG Chuan-Ping,JIANG Jing.Journal of Northeast Forestry University,2002,30(2):1-4王玉成,杨传平,姜静.东北林业大学学报,2002,30(2):1-4
9 WANG Ying-Fang,CHEN Yan-Lin,WANG Wen-Juan.Academic Journal of Guangdong College of Pharmacy,2017,33(1):18-22王颖芳,陈艳琳,王文娟.广东药科大学学报,2017,33(1):18-22
10 Jamalnasir H,Wagiran A,Shaharuddin N A,Samad A A.Aust.J.Crop Sci.,2013,7(7):911-916
11 WANG Su-Hui,XU Qian,XU Xiao,XU Ji-Chen.Journal of Jilin Agricultural University,2012,34(1):76-80王暑辉,徐倩,徐筱,徐吉臣.吉林农业大学学报,2012,34(1):76-80
12 LIU Jie,JIANG Jing-Yi,XU Ji-Chen.Molecular Plant Breeding,2014,12(3):554-561刘洁,江静怡,徐吉臣.分子植物育种,2014,12(3):554-561
13 Onrubia M,Moyano E,Bonfill M,Palazón J,Goossens A,CusidóR M.Plant Sci.,2011,181(3):282-287
14 YANG Xing-Xing,WANGRen-Cai,ZHANG Jia-Yin,LI Yan-Lin,QIN Yu,XIONG Xing-Yao.Journal of Hunan Agricultural University(Natural Sciences),2016,42(5):549-553杨星星,王仁才,张家银,李炎林,秦宇,熊兴耀.湖南农业大学学报(自然科学版),2016,42(5):549-553
15 GENG Xue-Jun,FENG Zi-Zhou,LI Xiao-Jiang,WANG Hai-Xia,FENG Lei,KANG Yu,WANG Ya-Nan,ZHANG MeiLin,WU Rui-Fang,ZHANG Ji-Xing.Journal of Inner Mongolia University for Nationalities(Natural Sciences),2014,29(6):653-655,719耿学军,冯紫洲,李晓江,王海霞,冯磊,康宇,王亚楠,张美琳,吴瑞芳,张继星.内蒙古民族大学学报(自然科学版),2014,29(6):653-655,719
16 Sambrook J,Russell D W,Editor.Molecular Cloning Experiment Guide,Translated by HUANG Pei-Tang,Beijing:Science Press,2002:343-356萨姆布鲁克J,拉塞尔D.W.主编,分子克隆实验指南,黄培堂主译.北京:科学出版社,2002:343-356
17 QIN Fang,WANG Jun-Min,HE Hai-Wang,HE Long-Fei,LI Chuang-Zhen,WEI Ben-Hui,HE Hu-Yi,GAN Xiu-Qin.Genomics and Applied Biology.,2009,28(4):755-759覃芳,王军民,何海旺,何龙飞,李创珍,韦本辉,何虎翼,甘秀芹.基因组学与应用生物学,2009,28(4):755-759
18 WU Hong-Qing,WANG Lei,GUO Wei,GAO Xiao-Xia,BAI Ling,ZHANG Wei-Min.Journal of Chinese Medicinal Materials,2013,36(7):1055-1059吴宏清,王磊,郭维,高晓霞,白玲,章卫民.中药材,2013,36(7):1055-1059
19 LOU Hui,ZHU Li-Cheng.Journal of Jing Gang Shan University(Natural Sciences),2017,38(6):35-37罗辉,朱立成.井冈山大学学报(自然科学版),2017,38(6):35-37
20 WU Yan-Fang,XU Hai-Ning,JIANG Xiang-Mei,WEN Qiang,XIONG Zhen-Yu.China Forestry Science and Technology,2013,27(4):75-79伍艳芳,徐海宁,江香梅,温强,熊振宇.林业科技开发,2013,27(4):75-79
21 ZHANG Yan-Mei,ZHOU Wen-Zhao,LI Jun-Feng.Molecular Plant Breeding,2010,8(1):201-208张燕梅,周文钊,李俊峰.分子植物育种,2010,8(1):201-208
2 ZHOU Hua,ZHU Qi,YANG Yan-Fang,YU Fa-Xin,QIU De-You.Plant Physiology Journal,2014,50(4):373-381周华,朱祺,杨艳芳,余发新,邱德有.植物生理学报,2014,50(4):373-381
3 GAO Ming-Bo,ZHANG Wei,LI Xing-Tai,RUAN Cheng-Jiang,FAN Sheng-Di.Chinese Journal of Biotechnology,2011,27(1):101-107高明波,张卫,李兴泰,阮成江,范圣第.生物工程学报,2011,27(1):101-107
4 LI Nai-Wei,SHU Xiao-Chun,HE Shu-Lan,JIANG Yu-Mei,XIA Bing,PENG Feng.Acta Botanica Boreali-Occidentalia Sinica,2010,30(12):2536-2541李乃伟,束晓春,何树兰,江玉梅,夏冰,彭峰.西北植物学报,2010,30(12):2536-2541
5 XIAO Lu,LIU Xian-Hu,LIU Ji-Sheng,LI Mei-Shan,XU Ming-Zi.Scientia Silvae Sinicae,2011,47(5):168-170肖璐,刘宪虎,刘继生,李美善,许明子.林业科学,2011,47(5):168-170
6 WU Qiong,DUAN Xiao-Qun,CHEN Xu,XIAO Pei-Gen.China Journal of Chinese Materia Medica,2012,37(24):3728-3733吴琼,段小群,陈旭,肖培根.中国中药杂志,2012,37(24):3728-3733
7 LI Yan-Lin,YANG Xing-Xing,ZHANG Jia-Yin,HUANG San-Wen,XIONG Xing-Yao.Acta Horticulturae Sinica,2014,41(4):735-745李炎林,杨星星,张家银,黄三文,熊兴耀.园艺学报,2014,41(4):735-745
8 WANG Yu-Cheng.,YANG Chuan-Ping,JIANG Jing.Journal of Northeast Forestry University,2002,30(2):1-4王玉成,杨传平,姜静.东北林业大学学报,2002,30(2):1-4
9 WANG Ying-Fang,CHEN Yan-Lin,WANG Wen-Juan.Academic Journal of Guangdong College of Pharmacy,2017,33(1):18-22王颖芳,陈艳琳,王文娟.广东药科大学学报,2017,33(1):18-22
10 Jamalnasir H,Wagiran A,Shaharuddin N A,Samad A A.Aust.J.Crop Sci.,2013,7(7):911-916
11 WANG Su-Hui,XU Qian,XU Xiao,XU Ji-Chen.Journal of Jilin Agricultural University,2012,34(1):76-80王暑辉,徐倩,徐筱,徐吉臣.吉林农业大学学报,2012,34(1):76-80
12 LIU Jie,JIANG Jing-Yi,XU Ji-Chen.Molecular Plant Breeding,2014,12(3):554-561刘洁,江静怡,徐吉臣.分子植物育种,2014,12(3):554-561
13 Onrubia M,Moyano E,Bonfill M,Palazón J,Goossens A,CusidóR M.Plant Sci.,2011,181(3):282-287
14 YANG Xing-Xing,WANGRen-Cai,ZHANG Jia-Yin,LI Yan-Lin,QIN Yu,XIONG Xing-Yao.Journal of Hunan Agricultural University(Natural Sciences),2016,42(5):549-553杨星星,王仁才,张家银,李炎林,秦宇,熊兴耀.湖南农业大学学报(自然科学版),2016,42(5):549-553
15 GENG Xue-Jun,FENG Zi-Zhou,LI Xiao-Jiang,WANG Hai-Xia,FENG Lei,KANG Yu,WANG Ya-Nan,ZHANG MeiLin,WU Rui-Fang,ZHANG Ji-Xing.Journal of Inner Mongolia University for Nationalities(Natural Sciences),2014,29(6):653-655,719耿学军,冯紫洲,李晓江,王海霞,冯磊,康宇,王亚楠,张美琳,吴瑞芳,张继星.内蒙古民族大学学报(自然科学版),2014,29(6):653-655,719
16 Sambrook J,Russell D W,Editor.Molecular Cloning Experiment Guide,Translated by HUANG Pei-Tang,Beijing:Science Press,2002:343-356萨姆布鲁克J,拉塞尔D.W.主编,分子克隆实验指南,黄培堂主译.北京:科学出版社,2002:343-356
17 QIN Fang,WANG Jun-Min,HE Hai-Wang,HE Long-Fei,LI Chuang-Zhen,WEI Ben-Hui,HE Hu-Yi,GAN Xiu-Qin.Genomics and Applied Biology.,2009,28(4):755-759覃芳,王军民,何海旺,何龙飞,李创珍,韦本辉,何虎翼,甘秀芹.基因组学与应用生物学,2009,28(4):755-759
18 WU Hong-Qing,WANG Lei,GUO Wei,GAO Xiao-Xia,BAI Ling,ZHANG Wei-Min.Journal of Chinese Medicinal Materials,2013,36(7):1055-1059吴宏清,王磊,郭维,高晓霞,白玲,章卫民.中药材,2013,36(7):1055-1059
19 LOU Hui,ZHU Li-Cheng.Journal of Jing Gang Shan University(Natural Sciences),2017,38(6):35-37罗辉,朱立成.井冈山大学学报(自然科学版),2017,38(6):35-37
20 WU Yan-Fang,XU Hai-Ning,JIANG Xiang-Mei,WEN Qiang,XIONG Zhen-Yu.China Forestry Science and Technology,2013,27(4):75-79伍艳芳,徐海宁,江香梅,温强,熊振宇.林业科技开发,2013,27(4):75-79
21 ZHANG Yan-Mei,ZHOU Wen-Zhao,LI Jun-Feng.Molecular Plant Breeding,2010,8(1):201-208张燕梅,周文钊,李俊峰.分子植物育种,2010,8(1):201-208