[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5 d)的紫花苜蓿(Medicago sativa L.)幼苗根、茎、叶组织中Actin2、GAPDH、MSC27、18S RNA、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,18S RNA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, bZIP, UBQ, PPPrep, Ms03_50f03 and Ms03_69f07)within different tissues(roots, stems and leaves)of alfalfa(Medicago sativa L.)through geNorm and NormFinder softwares, which were collected at different times(samples were collected after UV-B irradiation for 0, 1, 2 and 5 d). [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 UV-B 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 18S 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 geNorm. 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
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