JIA-2022-0201 多种胁迫和激素处理下番茄基因表达内参的转录组水平鉴定

doi:10.1016/j.jia.2022.07.051

摘要/Abstract

摘要： 番茄是研究果实发育和逆境响应的模式植物，基因表达分析是番茄研究中一项重要内容。定量PCR是一种应用广泛的基因表达分析技术，其中内参基因的选择可能会影响结果乃至结论的准确性。虽然番茄中已经有一些常用的参考基因，但研究表明，其中一些基因在不同组织或环境条件下的表达并不稳定。此外，目前从基因组水平鉴定和筛选番茄中内参基因的研究还很少。本研究从公开的转录测序数据中筛选出15个候选内参基因，并在胁迫和激素处理下分析了这些候选基因和7个传统使用的内参基因的表达稳定性。结果表明：一半以上的候选内参基因是番茄中的管家基因；不同的处理下最稳定表达的基因有所不同，在候选和传统使用的内参基因中，除了两个传统使用的Solyc04g009030和Solyc07g066610之外，大多数基因至少被推荐为首选内参基因一次。本研究不仅提供了番茄中的一些新的内参基因，而且还提供了不同环境条件下的首选内参基因，这有助于今后番茄的基因表达研究。我们的研究同时还表明，从转录组测序数据中挖掘稳定表达的基因是筛选qPCR分析内参基因的可靠方法。

Abstract:

Tomato (Solanum lycopersicum) is a model plant for research on fruit development and stress response, in which gene expression analysis is frequently conducted. Quantitative PCR (qPCR) is a widely used technique for gene expression analysis, and the selection of reference genes may affect the accuracy of results and even conclusions. Although there have been some frequently used reference genes in tomato, it has been shown that the expressions of some of these genes are not constant in different tissues and environmental conditions. Moreover, little information on genomic identification of reference genes is available in tomato. Here, we mined the publicly available transcriptional sequencing data and screened out fifteen candidate reference genes, and the expression stability of these candidate genes and seven traditionally used ones were evaluated under stress and hormone treatment. The results showed that over half of the selected candidate references were housekeeping genes in tomato cells. Among the candidate reference genes and the traditionally used ones, the most stably expressed genes varied under different treatments, and most of these genes were recommended as preferred reference genes at least once except Solyc04g009030 and Solyc07g066610, two traditionally used reference genes. This study provides some novel reference genes in tomato, and the preferred reference genes under different environmental stimuli, which may be useful for future research. Our study suggests that excavating stably expressed genes from transcriptome sequencing data is a reliable approach to screening reference genes for qPCR analysis.

Key words: tomato (Solanum lycopersicum) , gene expression , quantitative polymerase chain reaction (qPCR) , reference gene , expression stability

. JIA-2022-0201 多种胁迫和激素处理下番茄基因表达内参的转录组水平鉴定[J]. Journal of Integrative Agriculture, 2022, 21(11): 3216-3229.

DUAN Yao-ke, HAN Rong, SU Yan, WANG Ai-ying, LI Shuang, SUN Hao, GONG Hai-jun. Transcriptional search to identify and assess reference genes for expression analysis in Solanum lycopersicum under stress and hormone treatment conditions[J]. Journal of Integrative Agriculture, 2022, 21(11): 3216-3229.

参考文献

Alwine J C, Kemp D J, Stark G R. 1977. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proceedings of the National Academy of Sciences of the United States of America, 74, 5350–5354.
Andersen C L, Ledet-Jensen J, Ørntoft T. 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. Cancer Research, 64, 5245–5250.
Aslam M M, Rashid M A R, Siddiqui M A, Khan M T, Farhat F, Yasmeen S, Khan I A, Raja S, Rasool F, Sial M A, Zhao Y. 2022. Recent insights into signaling responses to cope drought stress in rice. Rice Science, 29, 105–117.
Bai Y, Kissoudis C, Yan Z, Visser R G F, Linden G V D. 2018. Plant behaviour under combined stress: Tomato responses to combined salinity and pathogen stress. The Plant Journal, 93, 781–793.
Barsalobres-Cavallari C F, Severino F E, Maluf M P, Maia I D G. 2009. Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions. BMC Molecular Biology, 10, 1–11.
Biondi A, Guedes R N C, Wan F H, Desneux N. 2018. Ecology, worldwide spread, and management of the invasive South American tomato pinworm, Tuta absoluta: Past, present, and future. Annual Review of Entomology, 63, 239–258.
Bustin S A, Benes V, Garson J A, Hellemans J, Huggett J F, Kubista M, Mueller R D, Nolan T, Pfaffl M W, Shipley G, Wittwer C T, Schjerling P, Day P J, Abreu M, Aguado B, Beaulieu J, Beckers A, Bogaert S, Browne J A, Carrasco-Ramiro F, et al. 2013. The need for transparency and good practices in the qPCR literature. Nature Methods, 10, 1063–1067.
Bustin S A, Nolan T. 2004. Pitfalls of quantitative real-time reverse transcription polymerase chain reaction. Journal of Biomolecular Techniques, 15, 155–166.
Cassol D, Cruz F P, Espindola K, Mangeon A, Müller C, Loureiro M E, Correa R L, Sachetto-Martins G. 2016. Identification of reference genes for quantitative RT-PCR analysis of microRNAs and mRNAs in castor bean (Ricinus communis L.) under drought stress. Plant Physiology and Biochemistry, 106, 101–107.
Cheng Y, Bian W, Pang X, Yu J, Ahammed G, Zhou G, Wang R, Ruan M, Li Z, Ye Q, Yao Z, Yang Y, Wan H. 2017. Genome-wide identification and evaluation of reference genes for quantitative RT-PCR analysis during tomato fruit development. Frontiers in Plant Science, 8, 1440.
Coker J S, Davies E. 2003. Selection of candidate housekeeping controls in tomato plants using EST data. BioTechniques, 35, 740–748.
Czechowski T, Stitt M, Altmann T, Udvardi M K, Scheible W R. 2005. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiology, 139, 5–17.
Dekkers B, Willems L, Bassel G, Bolderen-Veldkamp R, Ligterink W, Hilhorst H, Bentsink L. 2012. Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds. Plant and Cell Physiology, 53, 28–37.
Du L. 2013. Clone and function analysis of SlDP1 gene in tomato. MSc thesis, Chongqing University, China. (in Chinese)
Eisenberg E, Levanon E Y. 2013. Human housekeeping genes, revisited. Trends in Genetics, 29, 569–574.
Expósito-Rodríguez M, Borges A A, Borges-Pérez A, Pérez J A. 2008. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biology, 8, 131.
Fodor S P, Read J L, Pirrung M C, Stryer L, Lu A T, Solas D. 1991. Light-directed, spatially addressable parallel chemical synthesis. Science, 251, 767–773.
Gachon C, Mingam A, Charrier B. 2004. Real-time PCR: What relevance to plant studies? Journal of Experimental Botany, 55, 1445–1454.
Gao D, Kong F, Sun P, Bi G, Mao Y. 2018. Transcriptome-wide identification of optimal reference genes for expression analysis of Pyropia yezoensis responses to abiotic stress. BMC Genomics, 19, 251.
Gerszberg A, Hnatuszko-Konka K. 2017. Tomato tolerance to abiotic stress: A review of most often engineered target sequences. Plant Growth Regulation, 83, 175–198.
Gibson U E, Heid C A, Williams P M. 1996. A novel method for real time quantitative RT-PCR. Genome Research, 6, 995–1001.
Gong L, Yang Y, Chen Y, Shi J, Song Y, Zhang H. 2016. LbCML38 and LbRH52, two reference genes derived from RNA-Seq data suitable for assessing gene expression in Lycium barbarum L. Scientific Reports, 6, 37031.
González-Aguilera K, Saad C, Montes R, Alves-Ferreira M, Folter S. 2016. Selection of reference genes for quantitative real-time RT-PCR studies in tomato fruit of the genotype MT-Rg1. Frontiers in Plant Science, 7, 1386.
Gutierrez L, Mauriat M, Pelloux J, Bellini C, Wuytswinkel O V. 2008. Towards a systematic validation of references in real-time RT-PCR. The Plant Cell, 20, 1734–1735.
Habib S, Lwin Y Y, Li N. 2021. Down-regulation of SlGRAS10 in tomato confers abiotic stress tolerance. Genes, 12, 623.
Hoagland D R, Arnon D I. 1950. The water-culture method for growing plants without soil. California Agricultural Experiment Station Circular, 347, 1–32.
Hruz T, Wyss M, Docquier M, Pfaffl M W, Masanetz S, Borghi L, Verbrugghe P, Kalaydjieva L, Bleuler S, Laule O, Descombes P, Gruissem W, Zimmermann P. 2011. RefGenes: Identification of reliable and condition specific reference genes for RT-qPCR data normalization. BMC Genomics, 12, 156.
Huang X, Li S, Zhan A. 2019. Genome-wide identification and evaluation of new reference genes for gene expression analysis under temperature and salinity stresses in Ciona savignyi. Frontiers in Genetics, 10, 71.
Huggett J, Dheda K, Bustin S, Zumla A. 2005. Real-time RT-PCR normalisation; strategies and considerations. Genes and Immunity, 6, 279–284.
Huggett J, Dheda K, Bustin S A. 2006. Normalization. In: Dorak M T, ed., Real-Time PCR. Taylor & Francis Publishing, New York, the USA. pp. 83–91.
Jahan M S, Guo S, Sun J, Shu S, Wang Y, El-Yazied A A, Alabdallah N M, Hikal M, Mohamed M, Ibrahim M, Hasan M M. 2021. Melatonin-mediated photosynthetic performance of tomato seedlings under high-temperature stress. Plant Physiology and Biochemistry, 167, 309–320.
Lacerda A, Fonseca L N, Blawid R, Boiteux L S, Ribeiro S G, Brasileiro A. 2015. Reference gene selection for qPCR analysis in tomato-bipartite begomovirus interaction and validation in additional tomato-virus pathosystems. PLoS ONE, 10, e0136820.
Li T, Yuan W, Qiu S, Shi J. 2021. Selection of reference genes for gene expression analysis in Liriodendron hybrids’ somatic embryogenesis and germinative tissues. Scientific Reports, 11, 4957.
Lima L, Marchet C, Caboche S, Da Silva C, Istace B, Aury J M, Touzet H, Chikhi R. 2020. Comparative assessment of long-read error correction software applied to Nanopore RNA-sequencing data. Briefings in Bioinformatics, 21, 1164–1181.
Lin Y, Lin H, Cui H. 2018. Effect of exogenous H2S and H2O2 on the physiological characteristics of processing tomato seedlings under NaCl stress. Acta Agriculturae Boreali-Sinica, 22, 159–166. (in Chinese)
Long X, Liu Y, Rocheleau H, Ouellet T, Chen G. 2011. Identification and validation of internal control genes for gene expression in wheat leaves infected by strip rust. International Journal of Plant Breeding & Genetics, 5, 255–267.
Løvdal T, Lillo C. 2009. Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Analytical Biochemistry, 387, 238–242.
Lyndon R F, Francis D. 1992. Plant and organ development. Plant Molecular Biology, 19, 51–68.
Mandal S, Mallick N, Mitra A. 2009. Salicylic acid-induced resistance to Fusarium oxysporum f. sp. lycopersici in tomato. Plant Physiology and Biochemistry, 47, 642–649.
Martins P K, Mafra V, Souza W R D, Ribeiro A P, Vinecky F, Basso M F, Cunha B A D B, Kobayashi A K, Molinari H B C. 2016. Selection of reliable reference genes for RT-qPCR analysis during developmental stages and abiotic stress in Setaria viridis. Scientific Report, 6, 28348.
Mascia T, Santovito E, Gallitelli D, Cillo F. 2010. Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants. Molecular Plant Pathology, 11, 805–816.
Mele G, Hake S. 2003. Expression profiling of plant development. Genome Biology, 4, 215.
Müller O, Grau J, Thieme S, Prochaska H, Adlung N, Sorgatz A, Bonas U. 2015. Genome-wide identification and validation of reference genes in infected tomato leaves for quantitative RT-PCR analyses. PLoS ONE, 10, e0136499.
Nemeskéri E, Helyes L. 2019. Physiological responses of selected vegetable crop species to water stress. Agronomy, 9, 447.
Pfaffl M W, Tichopad A, Prgomet C, Neuvians T P. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using pair-wise correlations. Biotechnology Letters, 26, 509–515.
Pombo M A, Zheng Y, Fei Z, Martin G B, Rosli H G. 2017. Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato–Pseudomonas pathosystem. Scientific Report, 7, 44905.
Redman A M, Cipollini D F, Schultz J C. 2001. Fitness costs of jasmonic acid-induced defense in tomato, Lycopersicon esculentum. Oecologia, 126, 380–385.
Reid K E, Olsson N, Schlosser J, Peng F, Lund S T. 2006. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biology, 6, 27.
Rezzonico F, Nicot P C, Fahrentrapp J. 2018. Expression of tomato reference genes using established primer sets: Stability across experimental set-ups. Journal of Phytopathology, 166, 123–128.
Rivas S, Thomas C M. 2005. Molecular interactions between tomato and the leaf mold pathogen Cladosporium fulvum. Annual Review of Phytopathology, 43, 395–436.
Shinozaki K, Dennis E S. 2003. Cell signalling and gene regulation. Global analyses of signal transduction and gene expression profiles. Current Opinion in Plant Biology, 6, 405–409.
Shinozaki K, Yamaguchi-Shinozaki K. 2007. Gene networks involved in drought stress response and tolerance. Journal of Experimental Botany, 58, 221–227.
Silver N, Best S, Jiang J, Thein S L. 2006. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Molecular and Cell Biology, 7, 33.
Sims D, Sudbery I, Ilott N E, Heger A, Ponting C P. 2014. Sequencing depth and coverage: Key considerations in genomic analyses. Nature Reviews Genetics, 15, 121–132.
Smitha P K, Vishnupriyan K, Kar A S, Kumar M A, Bathula C, Chandrashekara K N, Dhar S K, Das M. 2019. Genome wide search to identify reference genes candidates for gene expression analysis in Gossypium hirsutum. BMC Plant Biology, 19, 405.
Sun W, Xu X, Zhu H, Liu A, Liu L, Li J, Hua X. 2010. Comparative transcriptomic profiling of a salt-tolerant wild tomato species and a salt-sensitive tomato cultivar. Plant and Cell Physiology, 6, 997–1006.
Tang X, Zhang N, Si H, Calderón-Urrea A. 2017. Selection and validation of reference genes for RT-qPCR analysis in potato under abiotic stress. Plant Methods, 13, 85.
Tong J, Hu M, Han B, Ji Y, Wang B, Liang H, Liu M, Wu Z, Liu N. 2021. Determination of reliable reference genes for gene expression studies in Chinese chive (Allium tuberosum) based on the transcriptome profiling. Scientific Reports, 11, 16558.
Vandesompele J, Preter K D, Pattyn F, Poppe B, Roy N V, Paepe A D, Speleman F. 2002. Accurate normalization of real-time quantitative RT-PCR data by geo-metric averaging of multiple internal control genes. Genome Biology, 3, 34.
Vinogradov A E. 2004. Compactness of human housekeeping genes: Selection for economy or genomic design? Trends in Genetics, 20, 248–253.
Vogelstein B, Kinzler K W. 1999. Digital PCR. Proceedings of the National Academy of Sciences of the United States of America, 96, 9236–9241.
Wan H, Zhao Z, Qian C, Sui Y, Malik A, Chen J. 2010. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Analytical Biochemistry, 399, 257–261.
Wang A M, Doyle M V, Mark D F. 1989. Quantitation of mRNA by the polymerase chain reaction. Proceedings of the National Academy of Sciences of the United States of America, 86, 9717–9721.
Wang X, Song Q, Liu Y, Brestic M, Yang X. 2022. The network centered on ICEs play roles in plant cold tolerance, growth and development. Planta, 255, 81.
Wang Z, Gerstein M, Snyder M. 2009. RNA-Seq: A revolutionary tool for transcriptomics. Nature Reviews Genetics, 10, 57–63.
Wieczorek P, Wrzesińska B, Obrępalska-Stęplowska A. 2013. Assessment of reference gene stability influenced by extremely divergent disease symptoms in Solanum lycopersicum L. Journal of Virological Methods, 194, 161–168.
Wong M L, Medrano J F. 2005. Real-time PCR for mRNA quantitation. Biotechniques, 39, 75–85.
Wu Y, Zhang C, Yang H, Lyu L, Li W, Wu W. 2021. Selection and validation of candidate reference genes for gene expression analysis by RT-qPCR in Rubus. International Journal of Molecular Sciences, 22, 10533.
Xie F, Xiao P, Chen D, Xu L, Zhang B. 2012. miRDeepFinder: A miRNA analysis tool for deep sequencing of plant small RNAs. Plant Molecular Biology, 80, 75–84.
Yang Z, Zhang R, Zhou Z. 2021. Identification and validation of reference genes for gene expression analysis in Schima superba. Genes (Basel), 12, 732.
Yin Z, Xie F, Michalak K, Zhang B, Zimnoch-Guzowska E. 2021. Reference gene selection for miRNA and mRNA normalization in potato in response to potato virus Y. Molecular and Cellular Probes, 55, 101691.
Zhang Y, Li H, Shang S, Meng S, Lin T, Zhang Y, Liu H. 2021. Evaluation validation of a qPCR curve analysis method and conventional approaches. BMC Genomics, 22, 680.
Zhang Z, Li C, Zhang J, Chen F, Gong Y, Li Y, Su Y, Wei Y, Zhao Y. 2020. Selection of the reference gene for expression normalization in Papaver somniferum L. under abiotic stress and hormone treatment. Genes (Basel), 11, 124.
Zhao J, Yang F, Feng J, Wang Y, Lachenbruch B, Wang J, Wan X. 2017. Genome-wide constitutively expressed gene analysis and new reference gene selection based on transcriptome data: A case study from poplar/canker disease interaction. Frontiers in Plant Science, 8, 1876.
Zhou Z, Cong P, Tian Y, Zhu Y, Yuan H. 2017. Using RNA-seq data to select reference genes for normalizing gene expression in apple roots. The Public Library of Science, 12, e0185288.
Zhu H, Ma Y, Guo Q. 2021. Expression stability of internal reference gene in response to Trichoderma polysporum infection in Avena fatua L. Current Genetics, 67, 909–918.