辣椒组织总RNA两种提取方法比较及qRT-PCR验证  

Comparison of Two Isolation Methods of Total RNA from Tissues of Capsicum Annuum and qRT-PCR Verification

作　　者：牛西强 罗潇云 胡能兵 黄先忠 NIU Xiqiang;LUO Xiaoyun;HU Nengbing;HUANG Xianzhong(College of Agriculture,Anhui Science and Technology University,Fengyang 233100,China)

机构地区：[1]安徽科技学院农学院,安徽凤阳233100

出　　处：《安徽科技学院学报》2021年第6期42-47,共6页Journal of Anhui Science and Technology University

基　　金：安徽科技学院学科带头人引进人才启动经费(NXYJ202001);安徽省大学生创新创业训练计划项目(S202010879303);安徽省科技攻关面上项目(1704g07020111);滁州市重点研究与开发计划项目(2020ZN001)。

摘　　要：目的:探索提取高质量的辣椒不同组织总RNA的方法,并通过实时荧光定量PCR(qRT-PCR)技术验证。方法:以1年生辣椒(Capsicum annuum)的根、下胚轴、茎、叶、顶端分生组织、花和果实为实验材料,比较改良Trizol Reagent试剂法和RNAprep Pure试剂盒法对辣椒组织总RNA的提取效果。以改良Trizol Reagent试剂法提取的辣椒不同组织总RNA为模板,反转录成cDNA的第一条链;通过RT-PCR和qRT-PCR,研究甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)的扩增和自我修剪基因(SELF-PRUNING,CaSP)的组织表达。结果:Trizol Reagent提取的总RNA,28 S和18 S条带较亮,OD_(260)/OD_(280)比值为1.82~2.07;RNAprep Pure提取的总RNA,28 S和18 S条带较暗,OD_(260)/OD_(280)比值为1.93~2.15;Trizol Reagent提取的总RNA能扩增出CaGAPDH基因片段,目的基因CaSP在不同组织中表达水平不同,在根和花中表达更高。结论:Trizol Reagent提取的总RNA,完整性和纯度均较好,得率也较高,能满足后续的分子实验要求。Trizol Reagent试剂法是提取高质量辣椒不同组织总RNA的较好方法。Objective:To explore the method of extraction of high quality total RNA from different tissues of Capsicum annuum,and verified by real-time fluorescent quantitative PCR(qRT-PCR)technology.Methods:Root,hypocotyl,stem,leaf,shoot apical meristem,flower and fruit of Capsicum annuum were used as experimental materials.Total RNA were extracted by the modified Trizol Reagent and RNAprep Pure methods,and their isolation effects were compared for total RNA extraction from different tissues of pepper.The total RNA extracted by modified Trizol Reagent methods was used as template to reversely transcribe the first strand of cDNA.RT-PCR and qRT-PCR were used to study the amplification of CaGAPDH(glyceraldehyde-3-phosphate dehydrogenase)gene and the tissue expression pattern of target gene CaSP(SELF-PRUNING).Results:The total RNA extracted by Trizol Reagent had bright of 28 S and 18 S,and the OD_(260)/OD_(280)ratio was 1.82-2.07.The total RNA extracted by RNAprep Pure had darker bands of 28 S and 18 S,and the OD_(260)/OD_(280)ratio was 1.93-2.15.The RNA extracted by Trizol Reagent can amplify CaGAPDH gene fragment.The expression level of target gene CaSP is different in tissues with higher expression levels in roots and flowers.Conclusion:The integrity and purity of total RNA extracted by Trizol Reagen were better and the yield was higher,which could meet the requirements of the following molecular experiments.The Trizol Reagent method is a better method for extracting total RNA from different tissues of pepper.

关 键 词：辣椒 总RNA 基因表达 QRT-PCR 自我修剪基因 

分 类 号：S641.3[农业科学—蔬菜学]