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.
作者
庞宏光
许建锋
张江红
张玉星
亓宝秀
PANG Hongguang;XU Jianfeng;ZHANG Jianghong;ZHANG Yuxing;QI Baoxiu(College of Horticulture,Hebei Agricultural University,Baoding 071001,Hebei,China;Beijing Collaborative Innovation Center for Eco-Environmental Improvement with Forestry and Fruit Trees,Beijing 100000,China)
出处
《果树学报》
CAS
CSCD
北大核心
2018年第A01期66-70,共5页
Journal of Fruit Science
基金
科技创新服务能力建设-科研基地-林果业生态环境功能提升协同创新中心(CEFF-PXM2018_014207_000024)
关键词
杜梨
不同组织
总RNA
纯度
浓度
Pyrus betulaefolia Bunge
Different tissues
Total RNA
Purity
Concentration
