Abstract:Abstract In the qRT-PCR analysis, selection of suitable reference genes is a prerequisite for the guarantee of accurate result. As the lack of genome information, the reference gene selection is particularly important for Chinese milk vetch (Astragalus sinicus). In this study, expression of seven commonly used housekeeping genes such as GAPDH, Cons7, ELF1B, Tubulin, Ubiquitin E2, Actin A and Actin 20 were assessed by qPCR in different tissues (roots,stems,leaves, flowers and pods) and different treatment (drought stress, waterlogging stress, salt stress, abscisic acid (ABA) stress) of Astragalus sinicus from NingBo. To determine the expression stability of these genes, qRT-PCR data were determined by three software tools (GeNorm, NormFinder and BestKeeper). The results showed that the highest expression stability candidate reference genes of Chinese milk vetch had many differences in the various experimental conditions. Cons7 could express stably under salt stress and waterlogging stress, and Tubulin, Actin 20, GAPDH were top ranked under different tissues, ABA treatment and drought stress respectively. In different tissues, Tubulin could be the best housekeeping gene. Cons7 was considered as the most appropriate housekeeping gene of Chinese milk vetch among abscisic acid treatment, salt stress and flooding stress, while GAPDH could be chosen as the best housekeeping gene under drought stress. The reference genes selected in this study will be helpful for improving the quality of gene expression studies under various stresses in Chinese milk vetch
[1] 孙清信, 陈坚, 张辉, 等. 2012. 紫云英ISSR引物的筛选及PCR反应体系的优化[J]. 植物遗传资源学报.13(5): 870-878. [2] 赵彩春, 李一星, 李友国, 等. 2011. 紫云英2个转脂蛋白基因在共生及重金属镉胁迫条件下的表达特征[J]. 华中农业大学学报. 30 (1): 18-22. [3] Andersen CL, Jensen JL, Orntoft TF. 2004. Normalization of real-time quantitative reverse transcription -PCR data: a model-based variance estimation approach to identify genes suited for normalization applied to bladder and colon cancer data sets [J]. Cancer research. 64 (15): 5245-5250.[4] Brunner AM, Yakovlev IA, Strauss SH. 2004. Validating internal controls for quantitative plant gene expression on studies [J]. BMC plant biology. 4: 14.[5] Chen DS, Li YG, Zhou JC. 2007. The symbiosis phenotype and expression patterns of five nodule-spec- ific genes of Astragalus sinicus under ammonium and salt stress conditions. Plant Cell Reports. 26 (8):1421-1430.[6] Dae J K, Dae S C, Sungchul C B, et al. 2007. Effects of soil selenium supplementation level on selenium contents of green tea leaves and milk vetch [J]. Journal of Food Science and Nutrition. 12(1): 35-39.[7] Hruz T, Wyss M, Docquier M, et al. 2011. RefGenes: identification of reliable and condition specific reference genes for RT-qPCR data normalization [J]. BMC Genomics 12: 156. [8] Jian B, Liu B, Bi Y, et al. 2008. Validation of internal control for gene expression study in soybean by quantitative real-time PCR [J]. BMC Molecular Biology. 9: 59.[9] Li Y, Zhou L, Li Y, et al. 2008. A nodule-specific plant cysteine proteinase, AsNODF32, is involved in nodule senescence and nitrogen fixation activity of the green manure legume Astragalus sinicus [J]. New Phytologist.180 (1):185-192.[10] Nakayama H. 2005. Characteristics of rice (Oryza sativa) growth, yield and soil nitrogen by cultivating Chinese milk vetch (Astragalus sinicus L.) as green manure [J]. Tohoku Agricultural Research. 58: 35-36.[11] Naomi A, Hideto U. 2009. Nitrogen dynamics in paddy soil applied with various 15 N-labelled green manures [J]. Plant and Soil. 322(1/2): 3-4.[12] Pfaffl MW, Tichopad A, Prgomet C, et al. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations [J]. Biotechnol Letters. 26 (6): 509-515.[13] Reddy DS1, Bhatnagar-Mathur P, Cindhuri KS, et al. 2013. Evaluation and validation of reference genes for normalization of quantitative real-time PCR based gene expression studies in peanut [J]. PLoS One. 8 (10): e78555.[14] Silver N, Best S, Jiang J, Thein SL.2006. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR [J]. BMC molecular biology. 7: 33.[15] Stürzenbaum SR, Kille P. 2001. Control genes in quantitative molecular biological techniques: the variability of invariance [J]. Comparative Biochemistry & Physiology Part A Molecular & Integrative Physiology. 130 (3): 281-289.[16] Taylor CM, Jost R, Erskine W, et al. 2016. Identifying Stable Reference Genes for qRT-PCR Normali- sation in Gene Expression Studies of Narrow-Leafed Lupin (Lupinus angustifolius L.) [J]. PLoS One.11 (2): e0148300.[17] Vandesompele J, De Preter K, Pattyn F, et al. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes [J].Genome Biology. 3(7): RESEARCH0034.[18] Wang J, Si Z, Li F, et al. 2015. A purple acid phosphatase plays a role in nodule formation and nitrogen fixation in Astragalus sinicus [J]. Plant Molecular Biology. 88 (6): 515-529.[19] Xie X, Huang W, Liu F, et al. 2013. Functional analysis of the novel mycorrhiza-specific phosphate transporter AsPT1 and PHT1 family from Astragalus sinicus during the arbuscular mycorrhizal symbiosis [J]. New Phytologist. 198 (3): 836-852.