Selection and Validation of Reference Genes for RT‐qPCR Analysis in Spinacia oleracea under Abiotic Stress

作者：Hao Xie, Bo Li, Yu‐Sun Chang, Xiaoyan Hou, Yue Zhang, Siyi Guo, Yuchen Miao, Quanhua Wang, Sixue Chen, Yinghua Su, Ying Li, Shaojun Dai · 发表于：BioMed Research International · 年份：2021 · DOI：10.1155/2021/4853632 · 被引用次数：41 · 研究领域：Molecular Biology Techniques and Applications、Animal Nutrition and Physiology、Plant Disease Resistance and Genetics

Reverse transcription quantitative real‐time polymerase chain reaction (RT‐qPCR) is an accurate and convenient method for mRNA quantification. Selection of optimal reference gene(s) is an important step in RT‐qPCR experiments. However, the stability of housekeeping genes in spinach ( Spinacia oleracea ) under various abiotic stresses is unclear. Evaluating the stability of candidate genes and determining the optimal gene(s) for normalization of gene expression in spinach are necessary to investigate the gene expression patterns during development and stress response. In this study, ten housekeeping genes, 18S ribosomal RNA ( 18S rRNA ), actin , ADP ribosylation factor ( ARF ), cytochrome c oxidase subunit 5C ( COX ), cyclophilin ( CYP ), elongation factor 1-alpha ( EF1α ), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), histone H3 ( H3 ), 50S ribosomal protein L2 ( RPL2 ), and tubulin alpha chain ( TUBα ) from spinach, were selected as candidates in roots, stems, leaves, flowers, and seedlings in response to high temperature, CdCl 2 , NaCl, NaHCO 3 , and Na 2 CO 3 stresses. The expression of these genes was quantified by RT‐qPCR and evaluated by NormFinder, BestKeeper, and geNorm. 18S rRNA , actin , ARF , COX , CYP , EF1α , GAPDH , H3 , and RPL2 were detected as optimal reference genes for gene expression analysis of different organs and stress responses. The results were further confirmed by the expression pattern normalized with different reference genes of two heat‐res...