|
|
Selection and Application of Reference Genes for qRT-PCR in Ustilago esculenta |
GE Xin-Tao, YIN Yu-Mei, GAO Li-Dan, HU Ying-Li, XIA Wen-Qiang, YE Zi-Hong, TANG Jin-Tian, ZHANG Ya-Fen* |
College of Life Sciences, China Jiliang University, Hangzhou 310018, China |
|
|
Abstract Wild rice stem is the expanded edible fleshy stem of Zizania latifolia, a Gamineous plant, when it is invaded by Ustilago esculenta. The selection of appropriate internal reference genes is important for analyzing the related gene expression profile in the study of the interaction between Zizania latifolia and Ustilago esculenta. In this study, the expression profiles of 8 candidate genes in U. esculenta were evaluated by qRT-PCR under different environmental conditions including 4 temperatures, 3 carbon source media, 5 developmental stages in vitro, and 5 time points during infection process. Data were analyzed using geNorm, NormFinder and BestKeeper software. In order to test the stability of the selected genes, the endoglucanase 1 gene (UeEgl1) was used as the test gene. The results showed that glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and α-tubulin expressed stably in all samples and were suitable for expression pattern analysis of U. esculenta. The results of this study could provide an effective correction tool for gene differential expression analysis of U. latifolia, ensure the accuracy and reliability of gene expression results, and provide reference for further analysis of the function of related genes in the process of interaction between U. latifolia and Z. latifolia.
|
Received: 19 June 2020
Published: 01 February 2021
|
|
Corresponding Authors:
* yfzhang@cjlu.edu.cn
|
|
|
|
[1] 郭得平, 李曙轩, 曹小芝. 1991. 茭白黑粉菌(Ustilago esculenta)某些生物学特性的研究[J]. 浙江农业大学学报, 17(1): 80-84. (Guo D P, Li S X, Cao X Z.1991. Study on some biological characteristics of Ustilago esculenta[J]. Journal of Zhejiang Agricultural University, 17(1): 80-84.) [2] 江解增, 韩秀芹, 曹碚生, 等. 2004. 茭白异地种植后共生黑粉菌差异性的研究[J]. 扬州大学学报, 25(1): 66-69. (Jiang J Z, Han X Q, Cao B S, et al.2004. Differences of Ustilago esculenta after Zizania latifolia grown in different places[J]. Journal of Yangzhou University, 25(1): 66-69.) [3] 王瑛, 陈亚娟, 丁莉萍, 等. 2016. 毛白杨不同组织器官稳定表达看家基因的筛选[J]. 植物生理学报, 52(8): 1312-1320. (Wang Y, Chen Y J, Ding L P, et al.2016. Validation of reference genes for gene expression analysis in different tissues of Populus tomentosa[J]. Plant Physiology Journal, 52(8): 1312-1320.) [4] 徐蝉, 张敬泽, 王晓清, 等. 2012. 美味黑粉菌冬孢子萌发条件的研究[J]. 长江蔬菜, 16: 104-107. (Xu C, Zhang J Z, Wang X Q, et al.2012. Research on factors influencing teliospore germination of Ustilago esculenta[J]. Journal of Changjiang Vegetables, 16: 104-107.) [5] 殷淯梅, 张雅芬, 曹乾超, 等. 2019. 茭白人工孕茭体系的建立及优化[J]. 农业生物技术学报, 27(8): 1498-1512. (Yin Y M, Zhang Y F, Cao Q C, et al.2019. Establishment and optimization of the artificial system for the formation of Jiaobai[J]. Journal of Agricultural Biotechnology, 27(8): 1498-1512.) [6] Andersen C L, Jensen J L, Orntoft T F.2004. Normalization of real-time quantitative reversetranscription-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. [7] Brefort T, Tanaka S, Neidig N, et al.2014. Characterization of the largest effector gene cluster of Ustilago maydis[J]. PLOS Pathogens, 10(7): e1003866. [8] Bustin S A, Benes V, Nolan T, et al.2005. Quantitative real-time RT-PCR-a perspective[J]. Journal of Molecular Endocrinology 34(3): 597-601. [9] Casquete R, Benito M J, Aranda E, et al.2019. Cyclopiazonic acid gene expression as strategy to minimizing mycotoxin contamination in cheese[J]. Fungal Biology, DOI: 10.1016/j.funbio.2019.06.011. [10] Chen Y X, Li B, Cen K, et al.2018. Diverse effect of phosphatidylcholine biosynthetic genes on phospholipid homeostasis, cell autophagy and fungal developments in Metarhizium robertsii[J]. Environmental Microbiology, 20(1): 293-304. [11] Chung K R, Tzeng D D.2004. Nutritional requirements of the edible gall-producing fungus Ustilago esculenta[J]. Journal of Biological Sciences 4(2): 246-252. [12] Dominguez R, Holmes K C.2011. Actin structure and function[J]. Annual Review of Biophysics, 40(1): 169-186. [13] Donofrio N M, Oh Y, Lundy R, et al.2006. Global gene expression during nitrogen starvation in the rice blast fungus, Magnaporthe grisea[J]. Fungal Genetics and Biology, 43(9): 605-617. [14] Ginzinger D G.2002. Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream[J]. Experimental Hematology, 30(6): 503-512. [15] Hellemans J, Mortier G, De Paepe A, et al.2007. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data[J]. Genome biology, 8(2): R19. [16] Kamper J, Kahmann R, Bolker M, et al.2006. Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis[J]. Nature, 444(7115): 97-101. [17] Llanos A, Francois J M, Parrou J L.2015. Tracking the best reference genes for RT-qPCR data normalization in filamentous fungi[J]. BMC Genomics, 16(1): 71. [18] Ma L S, Wang L, Trippel C, et al.2018. The Ustilago maydis repetitive effector Rsp3 blocks the antifungal activity of mannose-binding maize proteins[J]. Nature Communications, 9(1): 1-15. [19] Mosquera G, Giraldo M C, Khang C H, et al.2009. Interaction transcriptome analysis identifies Magnaporthe oryzae BAS1-4 as biotrophy-associated secreted proteins in rice blast disease[J]. Plant Cell, 21(4): 1273-1290. [20] Nygren K, Dubey M, Zapparata A, et al.2018. The mycoparasitic fungus Clonostachys rosea responds with both common and specific gene expression during interspecific interactions with fungal prey[J]. Evolutionary Applications, 11(6): 931-949. [21] Thellin O, Zorzi W, Lakaye B, et al.1999. Housekeeping genes as internal standards: Use and limits[J]. Journal of Biotechnology, 75(2-3): 291-295. [22] Omar C S, Bentley M A, Morieri G, et al.2016. Validation of reference genes for robust qRT-PCR gene expression analysis in the rice blast fungus Magnaporthe oryzae[J]. PLOS ONE, 11(8): e0160637. [23] Pathan E K, Ghormade V, Deshpande M V.2017. Selection of reference genes for quantitative real-time RT-PCR assays in different morphological forms of dimorphic zygomycetous fungus Benjaminiella poitrasii[J]. PLOS ONE, 12(6): 16. [24] Pfaffl M W, 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]. Biotechnology Letter, 26(6): 509-515. [25] Pickart C M, Eddins M J.2004. Ubiquitin: Structures, functions, mechanisms[J]. Biochimica et Biophysica Acta, 1695(1-3): 55-72. [26] Ruiz B, Chavez A, Forero A, et al.2010. Production of microbial secondary metabolites: Regulation by the carbon source[J]. Critical Reviews in Microbiology, 36(2): 146-167. [27] Schauwecker F, Wanner G, Kahmann R.1995. Filament-specific expression of a cellulase gene in the dimorphic fungus Ustilago maydis[J]. Biological Chemistry, 376(10): 617-625. [28] Sella L, Gazzetti K, Castiglioni C, et al.2016. The Fusarium graminearum Xyr1 transcription factor regulates xylanase expression but is not essential for fungal virulence[J]. Plant Pathology 65(5): 713-722. [29] Song L, Li T, Fan L, et al.2016. Identification and evaluation of reliable reference genes in the medicinal fungus Shiraia bambusicola[J]. Current Microbiology, 72(4): 444-449. [30] Soyer J L, El Ghalid M, Glaser N, et al.2014. Epigenetic control of effector gene expression in the plant pathogenic fungus Leptosphaeria maculans[J]. PLOS Genetics, 10(3): 19. [31] Tanaka S, Brefort T, Neidig N, et al.2014. A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize[J]. eLife, 3: e01355. [32] Tanaka S, Schweizer G, Rossel N, et al.2019. Neofunctionalization of the secreted Tin2 effector in the fungal pathogen Ustilago maydis[J]. Nature Microbiology, 4(2): 251-257. [33] 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): 0034.1-0034.11. [34] Wang Y, Wu J, Kim S G, et al.2016. Magnaporthe oryzae-secreted protein MSP1 induces cell death and elicits defense responses in rice[J]. Molecular Plant-Microbe Interactions, 29(4): 299-312. [35] Wang Z H, Yan N, Luo X, et al.2020. Gene expression in the smut fungus Ustilago esculenta governs swollen gall metamorphosis in Zizania latifolia[J]. Microbial Pathogenesis, 143: 104107. [36] Welchman R L, Gordon C, Mayer R J2005. Ubiquitin and ubiquitin-like proteins as multifunctional signals[J]. Nature reviews. Molecular Cell Biology, 6(8): 599-609. [37] Yang C D, Dang X, Zheng H W, et al.2017. Two Rab5 homologs are essential for the development and pathogenicity of the rice blast fungus Magnaporthe oryzae[J]. Frontiers in Plant Science, 8: 620. [38] Ye Z, Pan Y, Zhang Y, et al.2017. Comparative whole-genome analysis reveals artificial selection effects on Ustilago esculenta genome[J]. DNA Research, 24(6): 635-648. [39] You W Y, Liu Q A, Zou K Q, et al.2011. Morphological and molecular differences in two strains of Ustilago esculenta[J]. Current Microbiology, 62(1): 44-54. [40] Yu T, Wang Z H, Jin X C, et al.2014. Analysis of gene expression profiles in response to Sporisorium reilianum f. sp. zeae in maize (Zea mays L.)[J]. Electronic Journal of Biotechnology, 17(5): 230-237. [41] Zhang S P, Xiao Y N, Zhao J R, et al.2012. Digital gene expression analysis of early root infection resistance to Sporisorium reilianum f. sp. zeae in maize[J]. Molecular Genetics and Genomics, 288: 21-37. [42] Zhang Y F, Cao Q C, Hu P, et al.2017. Investigation on the differentiation of two Ustilago esculenta strains-implications of a relationship with the host phenotypes appearing in the fields[J]. BMC Microbiology, 17: 228. [43] Zhang Y F, Ge Q W, Cao Q C, et al.2018a. Cloning and characterization of two MAPK genes UeKpp2 and UeKpp6 in Ustilago esculenta[J]. Current Microbiology, 75(8): 1016-1024. [44] Zhang Y F, Liu H L, Cao Q C, et al.2018b. Cloning and characterization of the UePrf1 gene in Ustilago esculenta[J]. FEMS Microbiology Letters, 365(12): 10. [45] Zhang Y F, Wu M, Ge Q W, et al.2019a. Cloning and disruption of the UeArginase in Ustilago esculenta: Evidence for a role of arginine in its dimorphic transition[J]. BMC Microbiology, 19: 208. [46] Zhang Y F, Yin Y M, Hu P, et al.2019b. Mating-type loci of Ustilago esculenta are essential for mating and development[J]. Fungal Genetics and Biology, 125: 60-70. [47] Zhu W J, Wei W, Zhang S P, et al.2018. The phosphatome of medicinal and edible fungus Wolfiporia cocos[J]. Current Microbiology, 75(2): 124-131. |
|
|
|