张颖,陈婉婷,陈冉红,帅鹏,李明.杉木实时荧光定量PCR分析中内参基因的选择[J].林业科学研究,2019,32(2):65-72
杉木实时荧光定量PCR分析中内参基因的选择
Quantitative Real-time PCR Analysis of Cunninghamia lanceolata in the Selection of the Reference Genes
投稿时间:2018-01-09  修订日期:2018-11-28
DOI:10.13275/j.cnki.lykxyj.2019.02.010
中文关键词:  杉木  荧光定量  内参基因  BestKeeper
英文关键词:Cunninghamia lanceolata  fluorescence quantitative PCR  internal reference gene  BestKeeper
基金项目:国家自然科学基金青年基金项目(31500541);国家自然科学基金海峡联合基金项目(U1405211)
作者单位E-mail
张颖 福建农林大学林学院, 福建 福州 350002  
陈婉婷 福建农林大学林学院, 福建 福州 350002  
陈冉红 福建农林大学林学院, 福建 福州 350002  
帅鹏 福建农林大学林学院, 福建 福州 350002
国家林业和草原局杉木工程技术研究中心, 福建 福州 350002 
 
李明 福建农林大学林学院, 福建 福州 350002
国家林业和草原局杉木工程技术研究中心, 福建 福州 350002 
limingly@126.com 
摘要点击次数: 141
全文下载次数: 107
中文摘要:
      [目的]筛选适用于杉木不同组织荧光定量PCR分析的稳定内参基因,提高杉木实时荧光定量PCR试验准确性,为杉木体内各个基因表达分析提供合适内参基因。[方法]通过实时荧光定量PCR获取GAPDH、EF1α、18S rRNA、28S rRNA、UBQ、UBCActin 7个候选内参基因在杉木无性系的根、茎和叶中的溶解曲线与Ct值;利用软件geNorm、Normfinder和BestKeeper对候选内参基因的表达稳定性进行评价;通过分析2个纤维素合酶基因ClCesA1和ClCesA2在杉木不同组织部位的相对表达情况,对候选内参基因的稳定性进行验证。[结果]geNorm、Normfinder和BestKeeper分析结果均表明:在杉木根和茎中最稳定的内参基因是EF1αActin;geNorm、Normfinder结果显示杉木叶中稳定的内参基因是UBQEF1α,BestKeeper显示杉木叶中最稳定合适内参基因是<EF1αActin。以ActinEF1α作为内参基因,荧光定量PCR分析表明,ClCesA1和ClCesA2基因的相对表达量在杉木不同组织中均有所不同,ClCesA2的相对表达量在各个组织中均高于ClCesA1。以不同内参基因作为试验内参基因分析ClCesA1和ClCesA2的表达情况得到,ClCesA1和ClCesA2在各个组织中的相对表达量排序主要为茎 > 叶 > 根。[结论]7个候选内参基因中,杉木不同组织中稳定表达的为ActinEF1α,适合作为表达分析的内参基因;28S rRNA的表达稳定性低,不适合作为杉木定量PCR分析的理想内参基因。
英文摘要:
      [Objective] To improve the accuracy of real-time fluorescence quantitative PCR test and provide a suitable background reference for expression analysis of Chinese fir (Cunninghamia lanceolata), so as to select the stable internal control genes.[Method] The soluble curve and Ct value of seven candidate internal reference genes including GAPDH, EF1α, 18S rRNA, 28S rRNA, UBQ, UBC and Actin in roots, stems and leaves of Chinese fir clones were obtained by real-time fluorescence quantitative PCR. The expression stability of candidate reference genes was assessed by geNorm, Normfinder and BestKeeper. The expression stability of candidate reference genes was estimated by analyzing the relative expression of two cellulose synthase genes ClCesA1 and ClCesA2 in different tissues of Chinese fir.[Result] Three softwares, geNorm, Normfinder and BestKeeper, showed that the most stable internal reference genes expressed in Chinese fir roots and stems were EF1α and Actin. The software geNorm and Normfinder showed that the stable internal reference genes expressed in Chinese fir leaves were UBQ and EF1α. The software BestKeeper showed that the most stable and suitable internal reference genes for expression analysis are EF1α and Actin. Actin and EF1α as internal reference genes showed that the relative expression levels of ClCesA1 and ClCesA2 were different among the tissues of Chinese fir, and the relative expression level of ClCesA2 was higher than that of ClCesA1 in all tissues. According to expression analysis of different reference genes as test reference gene expression of ClCesA1 and ClCesA2, the relative expression levels of ClCesA1 and ClCesA2 were mainly stem > leaf > root.[Conclusion] Of the 7 candidate reference genes, Actin and EF1α are suitable as internal control genes for expression analysis due to stably expressed in different tissues of Chinese fir. 28S rRNA is not suitable as an ideal internal control gene for quantitative PCR analysis of Chinese fir as a result of the lower expression stability.
查看全文  查看/发表评论  下载PDF阅读器
关闭
京ICP备09064894号-3
版权所有:《林业科学研究》编辑部
地址:北京颐和园后中国林科院林业所 电话:010-62889680 E-mail:lykxyj@vip.163.com
技术支持:北京勤云科技发展有限公司
function PdfOpen(url){ var win="toolbar=no,location=no,directories=no,status=yes,menubar=yes,scrollbars=yes,resizable=yes"; window.open(url,"",win); } function openWin(url,w,h){ var win="toolbar=no,location=no,directories=no,status=no,menubar=no,scrollbars=yes,resizable=no,width=" + w + ",height=" + h; controlWindow=window.open(url,"",win); } &et=475F114B57537C4922F413D6F907F67E0F779D482576936E2235FEEC8AE7BC48403EBFA997FE6755BD7079314F93303F99CF96DDF65348C36E0B1E52EF3D2F4F37735413BA9806D0AF4E1EF2AD6BA94D1D61405952A25D730F5EBEC5F2FD3DBF1B55C6C7B8B71CB6208E8B8416A328BD&pcid=03F54A49DE00578AA0E5DDF5BC021AA7&cid=336C48A7E1AE975A&jid=578555B7B1AAB8ED64366E0DF87B2222&yid=B6351343F4791CA3&aid=&vid=&iid=0B39A22176CE99FB&sid=8BD23BD67BF01A5C&eid=AA76E167F386B6B3&fileno=20190210&flag=1&is_more=0"> var my_pcid="03F54A49DE00578AA0E5DDF5BC021AA7"; var my_cid="336C48A7E1AE975A"; var my_jid="578555B7B1AAB8ED64366E0DF87B2222"; var my_yid="B6351343F4791CA3"; var my_aid="";