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| Selection of Reference Gene in Magnolia denudata Under Salt Stress Using qRT-PCR |
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Abstract Abstract The selection of suitable reference genes can improve accuracy of gene expression analysis of qRT-PCR. In this study, the seedlings of Magnolia denudata were treated under salt stress in 4, 8, 12, 24, 48, 72 h. Root, steam and leaf tissues in different time were used as materials. 13 genes including ACTIN (actin), CYP (cyclophilin), EF-1α (elongationfactors-1α), GAPDH (glyceraldehyde-3-phosphat dehydrogenase), GBP (GTP binding protein), NAC (NAC domain protein), NADP (NADP-isocitrate dehydrogenase), TEF (translation elongation factors), UBC (ubiquitin-conjugating enzyme), UBQ (polyubiquitin protein), α-TUB (tubulin alpha), β-TUB (tubulin beta) and 18S (18S ribosomal RNA) were chosen as candidate reference genes. The specificity of the primers was investigated by agarose gel electrophoresis and melting curve. The softwares of geNorm, NormFinder and BestKeeper were used to analyze and screen optimum reference genes. Furthermore, two functional genes cellulose synthase-like D (CSLD) and endo-1,4-beta-d-glucanase (KOR) were selected to verify the reliability of the suitable reference genes. Electrophoresis results showed distinct PCR products with the expected size, amplification efficiency of primers were all between 95%~105%, and the single-peak melting curves further indicated the specificity of primers. GeNorm, NormFinder and BestKeeper comprehensive analysis showed that top 3 stable reference genes were GBP, UBQ and UBC, While 18S is the worst reference gene. Meanwhile, relative expression of CSLD and KOR in different tissues were analyzed by GBP, UBQ, UBC, the combination of top-stable genes (GBP, UBQ and UBC), the unstable gene 18S and GAPDH. As a result, the 2 target genes showed consistent expression profiles when normalized by 3 top-ranked reference genes and the combination, unstable genes 18S and GAPDH failed to standardize the expression data and caused deviation in the result. Therefore, GBP, UBQ and UBC could serve as stable reference genes for gene expression among different tissues in M. denudata under salt stress. The present study will provide an important reference basis for expression analysis of key genes of Magnolia in adverse situation
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Received: 15 December 2017
Published: 06 August 2018
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| Fund:The National Natural Science Foundation of China;the Public Welfare Forestry Industry Project of State Forestry Administration |
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