新疆野苹果果实总RNA提取方法的筛选

doi:10.13866/j.azr.2019.02.20

干旱区研究 ›› 2019, Vol. 36 ›› Issue (2): 437-443.doi: 10.13866/j.azr.2019.02.20

新疆野苹果果实总RNA提取方法的筛选

夏春兰^1,2,3, 闻志彬¹, 张玉芳¹

1.中国科学院新疆生态与地理研究所,中国科学院干旱区生物地理与生物资源重点实验室,新疆乌鲁木齐 830000;
2.中国科学院大学,北京 101408;
3.石河子大学生命科学学院,新疆石河子 832000

收稿日期:2018-04-24 修回日期:2018-08-30 发布日期:2025-10-18
通讯作者: 闻志彬. E-mail: zhibinwen@ms.xjb.ac.cn
作者简介:夏春兰(1996-),女,在读硕士生,专业方向为生物科学. E-mail:1416167406.com
基金资助:
国家重点研发计划(2016YFC0501505);中国科学院特色研究所主要服务项目(TSS-2015-014-FW-4-3)

Choosing of Methods for Total RNA Extraction from Malus sieversii Fruits in Xinjiang

XIA Chun-lan^1,2,3, WEN Zhi-bin¹, ZHANG Yu-fang¹

1. Key Laboratory of Biogeography and Biological Resources under Chinese Academy of Sciences,Xinjiang institute of Ecology and Geography,Chinese Academy of Sciences,Urumqi 830011,Xinjiang,China;
2. University of Chinese Academy of Sciences,Beijing 101408,China;
3. College of Life Science,Shihezi University,Shihezi 832000,Xinjiang,China

Received:2018-04-24 Revised:2018-08-30 Online:2025-10-18

摘要/Abstract

摘要： 由于新疆野苹果(Malus sieversii)果实富含多糖、多酚、可溶性果胶及其他次生代谢物质,导致果实的总RNA提取相对比较困难,质量较差。因此,本试验以不同发育时期的新疆野苹果的果实为材料,采用TaKaRa试剂盒、全氏金试剂盒、田伟等改良的CTAB法、本试验改良的CTAB法(一)和CTAB法(二)这5种方法来摸索提取野苹果果实的总RNA的有效方法。结果表明:2种试剂盒均无法从野苹果果实中提取出RNA;通过3种改良CTAB法提取的RNA,在琼脂糖凝胶上得到清晰的28S和18S条带,并且纯度均较高,OD₂₆₀/OD₂₈₀值在1.8~2.2之间、OD₂₆₀/OD₂₃₀值大于2.0。但是只有本试验改良CTAB法(一)获得较高的RNA产量,从5个不同发育时期的野苹果果实中提取的RNA平均值在116.79~474.76 μg·g^-1。改良CTAB法(一)通过延长LiCl沉淀时间,将-20 ℃放置15 min改为4 ℃放置过夜(时间不超过16 h),可以有效地除去RNA中的杂质污染,使得RNA充分沉淀。这种改良方法耗时相对较长,需要2 d,但是方法易于操作且提取成本较低。因此,改良CTAB法(一)是适用于提取野苹果果实(尤其是发育晚期)总RNA的有效方法,完全可以满足后期的分子生物学试验所需。

关键词: 新疆野苹果(Malus sieversii), 总RNA, 提取方法, 改良CTAB法

Abstract: Malus sieversii fruits are rich in polysaccharides,polyphenols,soluble pectin and other secondary metabolites. Therefore,it is relatively difficult to extract the total RNA from M.sieversii fruits. In order to choose an effective extraction method,five RNA extraction methods including the TaKaRa Kit,Trans Kit,improved CTAB method by Tian Wei,modified CTAB method I and CTAB method Ⅱwere compared using the fruits at five different developmental stages. Results showed that neither kits method was able to be used to extract the RNA from M.sieversii fruits. The quality of total RNA extracted with three modified CTAB methods was good,with two distinct electrophoresis bands of 28S and 18S rRNA,the OD₂₆₀/OD₂₈₀ values ranged from 1.8 to 2.2,and the OD₂₆₀/OD₂₃₀ value was higher than 2.0. But only the modified CTAB method I was an effective method to yield high quantity of total RNA. The average extraction values of fruits at five different developmental stages were ranged from 116.79 μg·g^-1 to 474.76 μg·g^-1. For the modified CTAB method I,the impurity in the RNA was effectively removed by extending LiCl precipitation time and placing extraction under 4 ℃ for overnight aging (not longer than 16 hours)instead of -20 ℃ for 15 min. Although this method took 2 days,the method was easy to operate,and the extraction cost was low. Therefore,the modified CTAB method I is an effective method for total RNA extraction from M.sieversii fruits (especially for the ripe fruits),which could be used in further molecular biology research.

Key words: Malus sieversii Roeml, total RNA, extraction method, improved CTAB method

夏春兰, 闻志彬, 张玉芳. 新疆野苹果果实总RNA提取方法的筛选[J]. 干旱区研究, 2019, 36(2): 437-443.

XIA Chun-lan, WEN Zhi-bin, ZHANG Yu-fang. Choosing of Methods for Total RNA Extraction from Malus sieversii Fruits in Xinjiang[J]. Arid Zone Research, 2019, 36(2): 437-443.

参考文献

[1] 张艳敏,冯涛,张春雨,等.新疆野苹果研究进展[J].园艺学报,2009,36(3):447-452.
[Zhang Yanmin,Feng Tao,Zhang Chunyu,et al.Advances in research of the Malus sieversii (Lebed.) Roeml.[J].Acta Horticulture Sinica,2009,36(3):447-452.]
[2] 宋益学.新疆野苹果的管理现状和保护措施[J].新疆林业,2006,30(6):34-35.
[Song Yixue.Management status and protection measures of Malus sieversii (Lebed.) Roeml.[J].Xinjiang Forestry,2006,30(6):34-35.]
[3] 李宇秀,张宏祥.死亡植株对新疆野苹果种群遗传多样性的影响[J].干旱区研究,2018,35(1):165-170.
[Li Yuxiu,Zhang Hongxiang.Effect of death individual on the genetic diversity of Malus sieversii population[J].Arid Zone Research,2018,35(1):165-170.]
[4] 姚玉新,赵玲玲,郝玉金,等.改良热硼酸法高效提取苹果果实RNA[J].果树学报,2005,22(6):141-144.
[Yao Yuxin,Zhao Linlin,Hao Yujin,et al.Effective extraction of total RNA in apple flesh with improved hot borate method[J].Journal of Fruit Science,2005,22(6):141-144.]
[5] 李宏,王新力.植物组织RNA提取的难点及对策[J].生物技术通报,1999,1(8):38-41.
[Li Hong,Wang Xili.The difficulties in the isolation of RNA from plant tissues and their resolving strategies[J].Biotechnology Bulletin,1999,1(8):38-41.]
[6] 刘芳,官春云.富含多酚类植物RNA提取的研究进展[J].作物研究,2015,29(1):91-95.
[Liu Fang,Guan Chunyun.Research progress on RNA extracting from plants rich in polyphenols[J].Crop Research,2015,29(1):91-95.]
[7] 杨亮,付丽娅,刘仲齐,等.富含多糖番茄果实组织中总RNA的有效提取方法[J].南开大学学报(自然科学版),2005,38(5):36-39.
[Yang Liang,Fu Liya,Liu Zhongqi,et al.An effective method for extracting total RNA from tomato fruit tissue rich in polysaccharides[J].Acta Scientiarum Naturalium Universitatis Nankaiensis,2005,38(5):36-39.]
[8] 郭秀莲,张正银,田萍,等.杜鹃花叶片总RNA的改良CTAB法提取[J].时珍国医国药,2010,21(1):29-31.
[Guo Xiulian,Zhang Zhengyin,Tian Ping,et al.Extraction of total RNA in Rhododendron leaf with improved CTAB method[J].Lishizhen Medicine and Materia Medica Research,2010,21(1):29-31.]
[9] 庄军平,苏箐,陈维信.一种从香蕉果实提取高质量RNA的方法[J].分子植物育种,2006,4(1):143-146.
[Zhuang Junping,Su Jing,Chen Weixin.An effective method for high-quality RNA isolation from banana fruit[J].Molecular Plant Breeding,2006,4(1):143-146.]
[10] 张卿,刘帅,邢宇,等.草莓果实总RNA提取方法的比较[J].中国农学通报,2015,31(31):146-149.
[Zhang Qing,Liu Shuai,Xing Yu,et al.Comparison of extraction methods of total RNA from strawberry fruits[J].Chinese Agricultural Science Bulletin,2015,31(31):146-149.]
[11] Lewinsohn E,Steele C L,Croteau R.Simple isolation of functional RNA from woody stems of gymnosperms[J].Plant Molecular Biology Reporter,1994,37(12):20-25.
[12] 田伟,田义轲,王彩虹,等.苹果组织总RNA提取方法的比较研究[J].青岛农业大学学报(自然科学版),2010,27(2):122-125.
[Tian Wei,Tian Yike,Wang Caihong,et al.Comparison of methods for total RNA extraction from apple tissues[J].Journal of Qingdao Agricultural University (Natural Science Edition),2010,27(2):122-125.]
[13] 杜凡.苹果冷胁迫下差异表达基因的筛选及新抗逆基因MdTLP7的功能鉴定[D].泰安:山东农业大学,2014.
[Du Fan.Identification of Differential Expression Genes under Cold Stress in Apple and Function Identification of a Novel Anti-Stress Gene MdTLP7[D].Tai'an:Shandong Agricultural University,2014.]
[14] Chang S,Puryear J,Cairney J.A simple and efficient method for isolating RNA from pine trees[J].Plant Molecular Biology Reporter,1993,59(11):113-116.
[15] 宋成秀,张利义,张彩霞,等.适合转录组测序的苹果果实RNA提取方法的筛选[J].中国果树,2015,2(23):47-49.
[Song Chengxiu,Zhang Liyi,Zhang Caixia,et al.Screening of RNA extraction methods suitable for transcriptome sequencing of apple fruit[J].China Fruits,2015,2(23):47-49.]
[16] Li M,Li D,Feng F,et al.Proteomic analysis reveals dynamic regulation of fruit development and sugar and acid accumulation in apple[J].Journal of Experimental Botany,2016,67(17):5145-5157.
[17] 李冬霞.苹果果糖积累相关基因MdTSTl和MdFK2启动子功能鉴定及其转录因子筛选[D].杨凌:西北农林科技大学,2016.
[Li Dongxia.Functional Identification of Fructose Accumulation Related Genes MdTSTl and MdFK2 Promoter and Screening of Transcription Factors in Apple Fruit[D].Yangling:Northwest Agriculture and Forestry University,2016.]
[18] 张晶.功能型苹果类黄酮合成相关基因表达分析及果实性状比较[D].泰安:山东农业大学,2014.
[Zhang Jing.Functional Expression Analysis of Flavonoid Synthesis Related Genes and Comparison of Fruit Characters in functional apple[D].Tai'an:Shandong Agricultural University,2014.]
[19] 闫述模,袁媛,杨滨,等.一种适用于不同发育期山楂果实总RNA提取的方法[J].中国实验方剂学杂志,2016,22(14):39-43.
[Yan Shumo,Yuan Yuan,Yang Bin,et al.Good method for isolating total RNA from crataegi fructus at different developmental stages[J].Chinese Journal of Experimental Traditional Medical Formulae,2016,22(14):39-43.]
[20] 马川.苹果、枣花孕育相关基因的克隆和功能表达研究[D].保定:河北农业大学,2010.
[Ma Chuan.Cloning and Functional Expression Analysis of Genes Related to Flower Initiation in Apple and Chinese Jujube Floral Bud[D].Baoding:Agricultural University of Hebei,2010.]
[21] 谭才邓,李静,邓毛程.神秘果果实总RNA提取方法比较研究[J].基因组学与应用生物学,2013,32(3):409-412.
[Tan Caideng,Li Jing,Deng Maocheng.Study on total RNA extraction methods from miracle fruit[J].Genomics and Applied Biology,2013,32(3):409-412.]
[22] 耿雪青.苹果果实cDNA文库的构建[D].北京:中国农业大学,2003.
[Geng Xueqing.Constructing cDNA Library of Apple Fruits[D].Beijing:China Agricultural University,2003.]
[23] 陈弟,符贤英,殷晓敏,等.几种果实总RNA提取方法的评价[J].广东农业科学,2007,11(17):30-33.
[Chen Di,Fu Xianying,Yin Xiaomin,et al.Assessment of several extraction methods of total RNA from fruits[J].Guangdong Agricultural Sciences,2007,11(17):30-33.]

新疆野苹果果实总RNA提取方法的筛选

Choosing of Methods for Total RNA Extraction from Malus sieversii Fruits in Xinjiang

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