UV-B胁迫下紫花苜蓿qRT-PCR内参基因的筛选  

Reference gene selection for qRT-PCR normalization in alfalfa under UV-B irradiation

作　　者：张薇[1] 崔为体[1] 段星亮 汪瑾[2] 沈文飚[1] 谢彦杰[1] 

机构地区：[1]南京农业大学生命科学学院,江苏南京210095 [2]南京农业大学生物学实验教学中心,江苏南京210095

出　　处：《南京农业大学学报》2015年第2期248-254,共7页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31200195;J1210056和J1310015);江苏省自然科学基金青年基金项目(BK2012364);教育部高等学校博士学科点专项科研基金项目(20120097120019);南京农业大学青年科技创新基金项目(KJ2012022)

摘　　要：[目的]在植物代谢通路关键调控基因表达的相关研究中,筛选各种条件下合适的内参基因至关重要。[方法]以UV-B为主要胁迫,运用荧光定量PCR技术以及geNorm和NormFinder软件,对不同取样时间(处理后0、1、2和5d)的紫花苜蓿(MedicagosativaL.)幼苗根、茎、叶组织中Actin2、GAPDH、MSC27、18SRNA、β-tublin、bZIP、UBQ、PPPrep、Ms03_50f03和Ms03_69f07等候选内参基因表达的稳定性进行研究。[结果]geNorm软件显示:紫花苜蓿幼苗根部UV-B胁迫下MSC27和UBQ的稳定值(M)较小,增加第3个内参基因后变异值(V)基本不变,表明选用2个内参基因即可;茎部则是MSC27和Actin2的M值较小,同时相应的V值也满足要求;而使用M值较小的Actin2和GAPDH为内参,在研究叶片基因表达时可以获得更加准确的结果,当增加第3个内参基因时,V值变大,反而影响结果。同时发现,18SRNA和β-tublin在根和茎叶中的稳定性均较差,因此不适宜作为内参基因使用。NormFinder的结果与上述结果相似,结果差异部分的原因可能是因为算法不同。[结论]在UV-B胁迫下,紫花苜蓿幼苗根部中MSC27和UBQ的表达较为稳定,而在茎部则以MSC27和Actin2为宜,使用Actin2和GAPDH为内参在研究叶片基因表达时可以获得更加准确的结果。本研究对UV-B胁迫下紫花苜蓿中关键基因的定量表达分析具有重要的实用价值。[Objectives]In order to acquire accurate results of the expression of genes during different metabolism processes in plants,it is critical to perform the selection of suitable reference genes under different conditions.[Methods]We analyze the stabilities of 10 reference genes（Actin2,GAPDH,MSC27,18S RNA,β-tublin,b ZIP,UBQ,PPPrep,Ms03_50f03 and Ms03_69f07） within different tissues（roots,stems and leaves） of alfalfa（Medicago sativa L.） through ge Norm and Norm Finder softwares,which were collected at different times（samples were collected after UV-B irradiation for 0,1,2 and 5d）.[Results]The results illustrated that MSC27 and UBQ were suitable for alfalfa roots analysis upon UV-B irradiation,as revealed by their lowest stability values.Furthermore,when another reference gene was added,corresponding variation（V） value（V3 /4） was not significantly altered when compared with that of V2 /3,which only contained two reference genes.These results further suggested that two reference genes were enough in alfalfa roots analysis under UVB stress.MSC27 and Actin2 was appropriated for the alfalfa stems analysis since the lowest M values were obtained.Meanwhile,the analysis of alfalfa leaves was advised to use Actin2 and GAPDH as internal genes under UV-B stress.The variation（V） value（V3 /4） was higher than that of V2 /3 when another reference gene was added,The 18 S RNA and β-tublin had the hightest M values in the analysis of all three alfalfa tissues,which suggested that they were not suitable for reference genes.The results analyzed by Normfinder were almost the same as that of ge Norm.The differences between these two softwares might be their different algorithms.[Conclusions]In alfalfa seedlings,MSC27 and UBQ were suitable in root analysis; MSC27 and Actin2 were appropriate in stem analysis; while we used Actin2 and GAPDH as internal genes to get more precise gene expression in leaf analysis.The results of the study have important practical value in the analysis of key genes under UV-B irr

关 键 词：紫花苜蓿 实时荧光定量PCR 内参基因 GENORM NORMFINDER 

分 类 号：Q781[生物学—分子生物学]