|
|
|
| Screening of Reference Genes for qRT-PCR Analysis in Sorghum (Sorghum bicolor) Under Low Nitrogen Stress |
| , , , |
|
|
|
|
Abstract Abstract Quantitative Real-time PCR is popularly used to study gene transcription levels in plants. Validation of candi-date reference genes is the key step to ensure accurate quantification. Nitrogen element assimilation and utilization play a critical role in sorghum (Sorghum bicolor) yield. The study was to select the most reliable reference genes for quantitative Real-time PCR (qRT-PCR) analysis of target sorghum genes under low nitrogen stress. We chose 7 commonly used housekeeping genes including Actin, ubiqutin conjugating enzyme (UBQ), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), UBQ10, eukaryotic translation initiation factor 2B (EIF2B), glyceraldehyde-3-phosphate dehydrogenase (sbGAPDH) and eukaryotic translation initiation factor 4a (EIF4a) to systematically assess their expression levels in leaves and roots of sorghum under low nitrogen stress by qRT-PCR. The results of primers specificity analysis showed that all the primers were in line with the requirements of the stability screening. The consequences of qRT- PCR indicated that the stability and expression level of candidate genes varied in the different tissues and under different treatments. The expression level of GAPDH was the highest in sorghum leaves, and that of UBQ10 was the highest in roots. We also analyzed and evaluated the stability of candidate reference genes by geNorm software. The results showed that GAPDH and EIF4a were the most suitable reference genes for normalizing target gene expression in sorghum leaves under low nitrogen stress, whereas sbGAPDH and EIF4a were recommended as the optimum combination in roots. In conclusion, this study has provided the basic data for expression analysis of target sorghum genes under low nitrogen stress and reference data for similar studies of other plants.
|
|
Received: 11 November 2016
Published: 02 May 2017
|
|
|
|
| 参考文献：Bustin S，Benes V，Nolan T，Pfaffl M. Quantitative real-time RT－PCR a perspective. J．Mol．Endocrinol.,2005,34(3): 597－601．Huggett J, Dheda K, Bustin S, Zumla A. Real-time RT-PCR normalisation; strategies and considerations.Genes & Immunity, 2005, 6(4):279-284.Pfaffl M W. Relative quantification. In: Dorak MT, ed. Real-time PCR. New York: International University Line. pp.2006: 63–82.Quackenbush J. Microarray data normalization and transformation. Nature Genetics, 2002, 32 suppl:496-501. Nolan T, Hands R E, Bustin S A. Quantification of mRNA using real-time RT-PCR. Nature Protocols, 2006, 1(3):1559-1582.Hu R B, Fan C M, Li H Y, Zhang Q, Fu Y F. Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. Bmc Molecular Biology, 2009, 10(1):93-93.Die J V, Román B, Nadal S, González-Verdejo Cl. Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta, 2010, 232(1):145-53.Warzybok A, Migocka M. Reliable reference genes for normalization of gene expression in cucumber grown under different nitrogen nutrition . PLoS One, 2013, 8(9):e72887. Manoli A,Sturaro A,Trevisan S, Quaggiotti S，Nonis A. Evaluation of candidate reference genes for qPCR in maize. Journal of Plant Physiology, 2012, 169(8): 807-815. Lovdal T, Lillo C. Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Analytical biochemistry,2009, 387(2):238-242.De Carvalho K, Bespalhok Filho J C, Dos Santos T B. Nitrogen starvation, salt and heat stress in coffee (Coffea arabica L.): identification and validation of new genes for qPCR normalization.Molecular Biotechnology, 2013, 53(3): 315-325.Xiao X L, Ma J B,Wang J R, et al. Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR . Frontiers in Plant Science, 2015,5:788.刘圆，王丽鸳，韦康，成浩，张芬，吴立赟，胡娟. 不同氮处理茶树实时定量 PCR 内参基因筛选和验证. 茶叶科学, 2016，36（1）：92-101. （Liu Y , Wang L Y , Wei K, CHeng H , ZHang F , Wu LY, Hu J. Screening and Validation of Reference Genes for Quantitative Real-time PCR Analysis in Tea Plant (Camellia sinensis) under Different Nitrogen Nutrition. Journal of Tea Science, 2016,36（1）：92-101.）袁伟,万红建 ,杨悦俭.植物实时荧光定量PCR 内参基因的特点及选择. 植物学报 ,2012,47(4):427–436. （Yuan W , Wan H J, Yang Y J. Characterization and Selection of Reference Genes for Real-time Quantitative RT-PCR of Plants. Chinese Bulletin of Botany ,2012, 47 (4): 427–436.）齐飞艳，胡 陶，彭镇华，高健.毛竹实时荧光定量ＰＣＲ内参基因的筛选及成花基因PheTFL1表达分析.西北植物学报,2013,33(1):0048-0052.（Qi F Y,Hu T,Peng ZH H,Gao J. Screening of reference genes used in qRT-PCR and expressionanalysis of PheTFL1 gene in Moso Bamboo. ActaBot.Boreal.Occident.Sin. 2013,33(1):0048-0052. ）Long X Y, Wang J R, Ouellet T,Rocheleau H, Wei Y M, Pu Z E, Jiang Q T, Lan X J, Zheng Y L. Genome-wide identification and evaluation of novel internal control genes for Q-PCR based transcript normalization in wheat. Plant Mol Biol ,2010,74:307–311.Maroufi A, van Bockstaele E, De Loose M. Validation of reference genes for gene expression analysis in chicory(Cichorium intybus) using quantitative Real-time PCR. BMC Molecular Biology, 2010,11: 15.Peidk E, Olsson N, Schlosser J. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol.,2006, 6:27-34.Islcandar H M, Simpson R S, Casu R E, etal. Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane. Plant Mol. Biol.Rep.,2004,22（4）:325-337.孙亚丽 张德辉 赵亮 夏聪聪 丑敏霞. 铜胁迫下天蓝苜蓿根组织实时定量PCR内参基因的选择. 农业生物技术学报,2014,22(10):1223-1231. （Sun Y L, Zhang D H, Zhao L, Xia C C, Chou M X.. Reference Gene Selection for Real- time Quantitative PCR in Black Medic (Medicago lupulina L.) Root Tissue Under Copper Stress. Journal of Agricultural Biotechnology, 2014,22(10):1223-1231.）Exgosito-Rodriguez M, Borges A A, Borges-Perez A. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol.,2008,8(1):131.Nicot N, Hausman J F, Hoffmann L. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress.J.Exp.Bot.,2005,56 (421):2907-2914.Nakayama T J, Rodrigues F A, Neumaier N, Marcelino- Guimar?es F C, Farias J R B, De Oliveira M C N, Borém A, De Oliveira A C B, Emygdio B M, Nepomuceno A L. Reference genes for quantitative real-time polymerase chain reaction studies in soybean plants under hypoxic conditions.Genet Mol Res,2014,13:860–871.Czechowski T, Stitt M, Altmann T．Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol.2005,139：5-17.Remans T,Smeets K, Opdenakker K. Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta,2008,227:1343-1349.Gutierrez L, Mauriat M, Guénin S, Pelloux J, Lefebvre J F, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C. The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT- PCR) analysis in plants. Plant Biotechnol J ,2008,6(6): 609–618.Hu R B, Fan C M, Li H Y, Zhang Q Z, Fu Y F. Evalua- tion of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol Biol ,2009,10:93.Tong Z G, Gao Z H, Wang F, Zhou J, Zhang Z. Selection of reliable reference genes for gene expression studies in peach using real time PCR. BMC Mol Biol,2009,10:71.魏毅东，陈玉，郭海萍,谢华安,,张建福,王宗华. 水稻缺素胁迫下实时荧光定量 R T -PCR 的内参基因的选择. 农业生物技术学报,2013,21(11):1302-1312. （Wei Y D, Chen Y, Guo H P, Xie H A, Zhang J F, Wang Z H.. Selection of Reference Genes for Real- time Quantitative RT- PCR in Rice (Oryza sativa L. ssp. japonica) under Nutrient Deficiency. Journal of Agricultural Biotechnology, 2013,21(11):1302-1312. ）Lim F H, Fakhrana I N, Rasid O A. Isolation and selection of reference genes for Ganoderma boninense gene expression study using quantitative Real-time PCR (qPCR). Journal of Oil Palm Research,2014,26(2):170-181.Xiao X L, Ma J B, Wang J R, Wu X, Li P. Validation of suitable reference genes for gene expression analysis in the halophyte Salicornia europaea by real-time quantitative PCR . Frontiers in Plant Science, 2015,5:788. |
|
|
|
