Special Issue:
昆虫合辑Plant Protection—Entomolgy
昆虫分子生物学合辑Insect Molecular Biology
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Stability evaluation of reference genes for real-time quantitative PCR normalization in Spodoptera frugiperda (Lepidoptera: Noctuidae) |
SHU Ben-shui1, YU Hai-kuo1, DAI Jing-hua1, XIE Zi-ge1, QIAN Wan-qiang2, LIN Jin-tian1 |
1 Guangzhou City Key Laboratory of Subtropical Fruit Trees Outbreak Control, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, P.R.China
2 Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120, P.R.China |
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摘要
本文选取Actin,EF1α,EF2,GAPDH,RPL3,RPL13,α-TUB和β-1-TUB基因作为候选内参基因;采用实时荧光定量PCR分析了8个候选内参基因在草地贪夜蛾不同龄期,6龄幼虫不同组织,幼虫在不同温度和饥饿处理等实验条件下的转录水平表达量;采用ΔCt,BestKeeper,geNorm,NormFinder和RefFinder对各个候选内参基因进行表达稳定性评估。GeNorm分析结果表明在本研究中不同实验条件下用于分析靶标基因转录水平表达量的最适内参基因个数均为2个。综合分析结果表明草地贪夜蛾不同发育阶段最稳定的内参基因为EF2和RPL13, 6龄幼虫不同组织中最稳定的内参基因为RPL13和β-1-TUB,不同温度处理下三龄幼虫中最稳定的内参基因为EF2和EF1α,饥饿处理条件下三龄幼虫中最稳定的内参基因为RPL3和EF1α,所有样本中较为稳定的内参基因为RPL13和EF1α。本研究为草地贪夜蛾不同实验条件下内参基因选择提供了参考,同时也有助于保证后续靶标基因转录水平表达研究的准确性。
Abstract Real-time quantitative PCR (qPCR) is a reliable and widely used technique for analyzing the expression profiles of target genes in different species, and reference genes with stable expressions have been introduced for the normalization of the data. Therefore, stability evaluation should be considered as the initial step for qPCR experiments. The fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is a polyphagous pest that consumes many plant species and seriously threatens corn production around the world. However, no studies thus far have examined the stability of reference genes in this pest. In this study, the expression profiles of the eight candidate reference genes of Actin, elongation factor 1 alpha (EF1α), elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L3 (RPL3), ribosomal protein L13 (RPL13), alpha-tubulin (α-TUB), and beta-1-tubulin (β-1-TUB) were obtained from S. frugiperda in different samples and the stability was evaluated by ΔCt, BestKeeper, geNorm, NormFinder, and RefFinder methods. The results of pairwise variation (V) calculated by GeNorm indicated two reference genes could be selected for normalization. Therefore, the combinations of the most stable reference genes for different experimental conditions of S. frugiperda were shown as follows: EF2 and RPL13 for developmental stages, RPL3 and β-1-TUB for larval tissue samples, EF2 and EF1α for the larval samples treated with different temperatures, RPL3 and EF1α for the larval samples under starvation stress, and RPL13 and EF1α for all the samples. Our results lay the foundation for the normalization of qPCR analyses in S. frugiperda and could help guarantee the accuracy of subsequent research.
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Received: 07 April 2020
Accepted:
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Fund: This work was financially supported by the fund from the Key-Area Research and Development Program of Guangdong Province, China (2020B020223004), the Innovation Team Project in Guangdong Provincial Department of Education (2017KCXTD018) and the Guangzhou Science and Technology Plan Projects, China (201704020190, 201805010008 and 201904010135). |
Corresponding Authors:
Correspondence LIN Jin-tian, Tel: +86-20-89003625, E-mail: linjtian@163.com
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About author: SHU Ben-shui, E-mail: shubenshui@126.com; |
Cite this article:
SHU Ben-shui, YU Hai-kuo, DAI Jing-hua, XIE Zi-ge, QIAN Wan-qiang, LIN Jin-tian.
2021.
Stability evaluation of reference genes for real-time quantitative PCR normalization in Spodoptera frugiperda (Lepidoptera: Noctuidae). Journal of Integrative Agriculture, 20(9): 2471-2482.
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Adeyinka O S, Tabassum B, Nasir I A, Yousaf I, Sajid I A, Shehzad K, Batcho A, Husnain T. 2019. Identification and validation of potential reference gene for effective dsRNA knockdown analysis in Chilo partellus. Scientific Reports, 9, 13629.
Babu S R, Kalyan R K, Sonika J, Balai C M, Mahla M K, Rokadia P. 2019. Report of an exotic invasive pest the fall armyworm, Spodoptera frugiperda (J.E. Smith) on maize in southern Rajasthan. Journal of Entomology and Zoology Studies, 7, 1296–1300.
Boaventura D, Bolzan A, Padovez F E, Okuma D M, Omoto C, Nauen R. 2020. Detection of a ryanodine receptor target-site mutation in diamide insecticide resistant fall armyworm, Spodoptera frugiperda. Pest Management Science, 76, 47–54.
Bustin S A, Benes V, Garson J, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl M W, Shipley G, Wittwer C T, Schjerling P, Day P J, Abreu M, Aguado B, Beaulieu J F, 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.
Caridi C P, Plessner M, Grosse R, Chiolo I. 2019. Nuclear actin filaments in DNA repair dynamics. Nature Cell Biology, 21, 1068–1077.
Chandra G S, Asokan R, Manamohan M, Kumar N K, Sita T. 2014. Evaluation of reference genes for quantitative real-time PCR normalization in cotton bollworm Helicoverpa armigera. Molecular Biology, 48, 813–822.
Chen Z S, Han N N, Li J H, Wan H, Huang G H. 2017. Selection and validation of reference genes for RT-qPCR analysis in Sf9 cell line infected by Heliothis virescens ascovirus 3h (HvAV-3h). Journal of Asia-Pacific Entomology, 20, 463–468.
Concha C, Edman R M, Belikoff E J, Schiemann A H, Carey B, Scott M J. 2012. Organization and expression of the Australian sheep blowfly (Lucilia cuprina) hsp23, hsp24, hsp70 and hsp83 genes. Insect Molecular Biology, 21, 169–180.
Ehara H, Kujirai T, Fujino Y, Shirouzu M, Kurumizaka H, Sekine S I. 2019. Structural insight into nucleosome transcription by RNA polymerase II with elongation factors. Science, 363, 744–747.
Feldmann F, Rieckmann U, Winter S. 2019. The spread of the fall armyworm Spodoptera frugiperda in Africa: What should be done next? Jouranl of Plant Diseases and Protection, 126, 97–101.
Feng Z, Zhang L, Wu Y, Wang L, Xu M, Yang M, Li Y, Wei G, Chou M. 2019. The Rpf84 gene, encoding a ribosomal large subunit protein, RPL22, regulates symbiotic nodulation in Robinia pseudoacacia. Planta, 250, 1897–1910.
Fu W, Xie W, Zhang Z, Wang S, Wu Q, Liu Y, Zhou X, Zhou X, Zhang Y. 2013. Exploring valid reference genes for quantitative real-time PCR analysis in Plutella xylostella (Lepidoptera: Plutellidae). International Journal of Biological Science, 9, 792–802.
Garcia A G, Ferreira C P, Godoy W A C, Meagher R L. 2019. A computational model to predict the population dynamics of Spodoptera frugiperda. Journal of Pest Science, 92, 429–441.
Goergen G, Kumar P L, Sankung S B, Togola A, Tamò M. 2016. First report of outbreaks of the fall armyworm Spodoptera frugiperda (J E Smith) (Lepidoptera, Noctuidae), a new alien invasive pest in West and Central Africa. PLoS ONE, 11, e0165632.
Gouin A, Bretaudeau A, Nam K, Gimenez S, Aury J M, Duvic B, Hilliou F, Durand N, Montagné N, Darboux I, Kuwar S, Chertemps T, Siaussat D, Bretschneider A, Moné Y, Ahn S J, Hänniger S, Grenet A G, Neunemann D, Maumus F, et al. 2017. Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges. Scientific Reports, 7, 11816.
Hellemans J, Vandesompele J. 2014. Selection of reliable reference genes for RT-qPCR analysis. Methods in Molecular Biology, 1160, 19–26.
Hu Z, Deng Y C, Zhang X L, Tang P P, Sun W J, Li G H, Yu Q, Yao Q. 2018. Selection and validation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) in silkworm infected with Bombyx mori bidensovirus. Biologia, 73, 897–906.
Jiang N J, Wang C Z. 2019. Progress in sex pheromone communication of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae). Acta Entomologica Sinica, 62, 993–1002. (in Chinese)
Kakumani P K, Malhotra P, Mukherjee S K, Bhatnagar R K. 2014. A draft genome assembly of the army worm, Spodoptera frugiperda. Genomics, 104, 134–143.
Kang Z W, Liu F H, Tian H G, Zhang M, Guo S S, Liu T X. 2017. Evaluation of the reference genes for expression analysis using quantitative real-time polymerase chain reaction in the green peach aphid, Myzus persicae. Insect Science, 24, 222–234.
Kariyanna B, Prabhuraj A, Asokan R, Babu P, Jalali S K, Venkatesan T, Gracy R G, Mohan M. 2020. Identification of suitable reference genes for normalization of RT-qPCR data in eggplant fruit and shoot borer (Leucinodes orbonalis Guenee). Biologia, 75, 289–297.
Li K, Xu N, Yang Y J, Zhang J H, Yin H. 2018. Identification and validation of reference genes for RT-qPCR normalization in Mythimna separata (Lepidoptera: Noctuidae). Biomed Research International, 2018, 1828253.
Ling D, Salvaterra P M. 2011. Robust RT-qPCR data normalization: validation and selection of internal reference genes during post-experimental data analysis. PLoS ONE, 6, e17762.
Liu G, Qiu X, Cao L, Zhang Y, Zhan Z, Han R. 2016. Evaluation of reference genes for reverse transcription quantitative PCR studies of physiological responses in the ghost moth, Thitarodes armoricanus (Lepidoptera, Hepialidae). PLoS ONE, 11, e0159060.
Liu Y, Xiao H, Mei Y, Yang Y, Ye X, Chen A, Li F. 2019. Evolutionary analysis of chemoreception related gene families of Spodoptera frugiperda. Journal of Environmental Entomology, 41, 718–726.
Lü J, Chen S, Guo M, Ye C, Qiu B, Wu J, Yang C, Pan H. 2018a. Selection and validation of reference genes for RT-qPCR analysis of the ladybird beetle Henosepilachna vigintioctomaculata. Frontiers in Physiology, 9, 1614.
Lü J, Yang C, Zhang Y, Pan H. 2018b. Selection of reference genes for the normalization of RT-qPCR data in gene expression studies in insects: A systematic review. Frontiers in Physiology, 9, 1560.
Mei Y, Yang Y, Ye X, Xiao H, Li F. 2019. Evolutionary analysis of detoxification gene families of Spodoptera frugiperda. Journal of Environmental Entomology, 41, 727–735.
Montezano D G, Specht A, Sosa-Gómez D R, Roque-Specht V F, Sousa-Silva J C, Paula-Moraes S V, Peterso J A, Hunt T E. 2018. Host plants of Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Americas. African Entomology, 26, 286–301.
Nandakumar S, Ma H, Khan A S. 2017. Whole-genome sequence of the Spodoptera frugiperda Sf9 insect cell line. Genome Announcement, 5, e00829–e00846.
Pan H, Yang X, Bidne K, Hellmich R L, Siegfried B D, Zhou X. 2015. Selection of reference genes for RT-qPCR analysis in the monarch butterfly, Danaus plexippus (L.), a migrating bio-indicator. PLoS ONE, 10, e0129482.
Pashley D P. 1986. Host-associated genetic differentiation in fall armyworm (Lepidoptera, Noctuidae): a sibling species complex? Annals of the Entomological Society of America, 79, 898–904.
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.
Reall T, Kraus S, Goodman C L, Ringbauer Jr J, Geibel S, Stanley D. 2019. Next-generation cell lines established from the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro Cellular & Developmental Biology (Animal), 55, 686–693.
Rodrigues T B, Khajuria C, Wang H, Matz N, Cunha Cardoso D, Valicente F H, Zhou X, Siegfried B. 2014. Validation of reference housekeeping genes for gene expression studies in western corn rootworm (Diabrotica virgifera virgifera). PLoS ONE, 9, e109825.
Shakeel M, Rodriguez A, Tahir U B, Jin F. 2018. Gene expression studies of reference genes for quantitative real-time PCR: An overview in insects. Biotechnology Letters, 40, 227–236.
Shu B, Zhang J, Cui G, Sun R, Sethuraman V, Yi X, Zhong G. 2018. Evaluation of reference genes for real-time quantitative PCR analysis in larvae of Spodoptera litura exposed to azadirachtin stress conditions. Frontiers in Physiology, 9, 372.
Shu B, Zhang J, Zeng J, Cui G, Zhong G. 2019. Stability of selected reference genes in Sf9 cells treated with extrinsic apoptotic agents. Scientific Reports, 9, 14147.
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 Biology, 7, 33.
Sun M, Lu M X, Tang X T, Du Y Z. 2015. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae). PLoS ONE, 10, e0115979.
Sun X, Hu C, Jia H, Wu Q, Shen X, Zhao S, Jiang Y, Wu K. 2021. Case study on the first immigration of fall armyworm Spodoptera frugiperda invading into China. Journal of Integrative Agriculture, 20, 664–672.
Vandesompele, J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3, RESEARCH0034.
Vantaku V, Chauhan V K, Dhania N K, Senthilkumaran B, Dutta-Gupta A. 2019. Validation of reference gene(s) for quantitative gene expression profiling using rice moth, Corcyra cephalonica as a model. Gene Reports, 14, 25–29.
Wang Z, Meng Q, Zhu X, Sun S, Liu A, Gao S, Gou Y. 2020. Identification and evaluation of reference genes for normalization of gene expression in developmental stages, sexes, and tissues of Diaphania caesalis (Lepidoptera, Pyralidae). Journal of Insect Science, 20, 6.
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.
You Y, Xie M, Vasseur L, You M S. 2018. Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.) Genome, 61, 349–358.
Zhang B Z, Liu J J, Chen X L, Yuan G H. 2018. Selection and evaluation of reference genes for gene expression using quantitative real-time PCR in Mythimna separata Walker (Lepidoptera: Noctuidae). Entomological Research, 48, 390–399.
Zhang S, An S, Li Z, Wu F, Yang Q, Liu Y, Cao J, Zhang H, Zhang Q, Liu X. 2015. Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in Helicoverpa armigera (Lepidoptera: Noctuidae). Gene, 555, 393–402.
Zheng L, Zhang Z J, Zhang J M, Li X W, Huang J, Lin W C, Li W D, Li C R, Lu Y B. 2019. Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) sex-dimorphic development. Journal of Integrative Agriculture, 18, 854–864.
Zhou C, Wang L, Price M, Li J, Meng Y, Yue B S. 2020. Genomic features of the fall armyworm (Spodoptera frugiperda) (J.E. Smith) yield insights into its defense system and flight capability. Entomological Research, 50, 100–112.
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