Identification and Verification of Somatic Embryogenesis mRNA and miRNA qPCR Reference Genes in Garlic (Allium sativum)
LI Meng-Qian, LIU Min, ZHANG Meng, BAI Yun-He, LI Ping, WEI Han-Yu, ZHOU Rong, JIANG Fang-Ling, WU Zhen*
Key Laboratory of Biology and Germplasm Enhancement of Horticultural Crops in East China, Ministry of Agriculture, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
Abstract：Garlic (Allium sativum) is an important vegetable crop for asexual reproduction, and the molecular mechanism research of somatic embryogenesis is still relatively weak. Selection of the stable reference genes will help clarify the differential expression of genes and miRNAs in somatic cell formation, help lay the foundation for the research to reveal the mechanism of garlic somatic embryogenesis. To select the most stable reference genes for mRNA and miRNA qPCR detection system during garlic somatic embryogenesis, this study chose 7 mRNA candidate reference genes, including the actin (AsACTIN), glyceraldehyde-3-phosphate dehydrogenase (AsGAPDH), 18S ribosomal RNA (As18S rRNA), polyubiquitinase (AsUBQ), α-tubulin (AsTUA), transcription factors LATE ELONGATED HYPOCOTYL (AsLHY) and Brassinosteroid resistant 1 (AsBES1). 5 miRNA candidate reference genes, including AsU6snRNA, As5.8S rRNA, AsmiR159a-1, AsmiR168a and AsmiR168a-5p. qPCR technology was used to detect their Ct value at different stages of somatic embryo development in different genotypes, explants, and 2,4-D concentrations. Delta CT, BestKeeper, NormFinder, and GeNorm were applied to analyze the expression stability of candidate reference genes. The results were combined with RefFinder to evaluate the expression stability of candidate reference genes comprehensively. The results showed that for mRNA qPCR expression analysis, AsACTIN was the most suitable reference gene for different genotypes. AsBES1 was the most stable reference gene under different explants and different 2,4-D concentrations. The most stable reference gene in all samples was AsBES1. For miRNA, the most stable reference gene in different genotypes was AsmiR159a-1, AsmiR168a-5p was the most stable reference gene in different explants, and As5.8S rRNA was the most stable reference gene under different 2,4-D concentration treatments. The ideal reference gene in all samples was AsU6 snRNA. Using the 7 mRNA candidate reference genes and 5 miRNA candidate reference genes, plant hormone related transcription factors myelocytomatosis protein 2 (AsMYC2) and AsmiR167 were analyzed by qPCR, which were differentially expressed during garlic somatic embryogenesis. The results showed that AsBES1 and AsmiR168a-5p, which were the most stable reference gene during garlic somatic embryogenesis of different explants, were used as reference gene, the expression trends of AsMYC2 and AsmiR167d-1 were consistent with the sequencing data. When other genes (miRNA) were used as controls, the gene expression trends (miRNA) differed, further confirming the reliability of reference genes. This study provides supports for accurately detecting genes and miRNAs during garlic somatic embryogenesis, it is helpful to further study the mechanism of somatic embryogenesis in garlic.
[1] 陈敏敏, 张茹佳, 查倩, 等. 2018. 百合体胚诱导、发育及不同组织实时定量PCR内参基因筛选[J]. 分子植物育种, 16(15): 4982-4990. (Chen M M, Zhang R J, Cha Q, et al.2018. Induction and development of Lily somatic embryo and reference genes screening for qRT-PCR in different tissues[J]. Molecular Plant Breeding, 16(15): 4982-4990.) [2] 李杉, 邢更妹, 崔凯荣, 等. 2001. 植物体细胞胚发生中ATP酶活性时空分布动态与内源激素的变化[J]. 植物学通报, (03): 308-317. (Li S, Xing G M, Cui K R, et al. 2001. ATPase activity and endogenous hormone changes during plant somatic embryogenesis[J]. Chinese Bulletin of Botany, (03): 308-317.) [3] 刘政. 2015. 柑橘珠心胚起始转录组分析及体细胞胚发生相关基因CsFUS3功能鉴定[D]. 博士学位论文, 华中农业大学, 导师: 郭文武, pp. 3-12. (Liu Z.2015. Transcriptional analysis of citrus nucellar embryo initiation and functional characterization of CsFUS3 gene preferentially expressed during somatic embryogenesis[D]. Thesis for Ph.D., Huazhong Agricultural University, Supervisor: Guo W W, pp. 3-12.) [4] 刘敏. 2018. 活性氧介导的大蒜试管苗玻璃化发生机制[D]. 博士学位论文, 南京农业大学, 导师: 吴震, pp. 72-76. (Liu M.2018. Mechanism of hyperhydricity mediated by reactive oxygen species of garlic plantlets in vitro[D]. Thesis for Ph. D., Nanjing Agricultural University, Supervisor: Wu Z, pp. 72-76.) [5] 刘世琦. 2007. 蔬菜栽培学简明教程[M]. 化学工业出版社, 北京. pp. 173. (Liu S Q.2007. Cornelii Cursu Vegetable Cultivation[M]. Chemical Industry Press. Beijing. pp. 173.) [6] 刘晓雪, 程智慧. 2013. 大蒜种质超低温保存及脱毒技术研究进展[J]. 中国蔬菜, (02): 12-19. (Liu X X, Cheng Z H. 2013. Research progress on cryopreservation of garlic germplasm and its virus eradication technique[J]. China Vegetables, (02): 12-19.) [7] 林玉玲, 赖钟雄. 2011. 龙眼胚性愈伤组织miR398a前体的克隆及其在龙眼体胚发生过程中的表达分析[J]. 热带作物学报, 32(04): 632-639. (Lin Y L, Lai Z X.2011. Cloning of miR398a precursor gene from embryogenic callus and its expression analysis during somatic embryogenesis in Longan[J]. Chinese Journal of Tropical Crops, 32(04): 632-639.) [8] 吕运舟, 董筱昀, 黄利斌. 2019. 黄山栾树实时荧光定量PCR内参基因的筛选[J]. 分子植物育种, 17(02): 553-560. (Lv Y Z, Dong X Y, Huang L B.2019. The screening of reference genes for real-time fluorescent quantitative PCR of Koelreuteria bipinnata[J]. Molecular Plant Breeding, 17(02): 553-560.) [9] 马璐琳, 崔光芬, 王祥宁, 等. 2019. 西南鸢尾花色变异实时定量PCR内参基因的筛选与验证[J]. 核农学报, 33(09): 1707-1716. (Ma L L, Cui G F, Wang X N, et al.2019. Selection and validation of reference genes for quantitative real-time PCR analysis in Iris bulleyana Dykes during flower color variation[J]. Journal of Nuclear Agricultural Sciences, 33(09): 1707-1716.) [10] 齐香玉, 陈双双, 冯景, 等. 2020. 茉莉花实时荧光定量PCR内参基因的筛选与验证[J]. 华北农学报, 35(06): 22-30. (Qi X Y, Chen S S, Feng J, et al.2020. Selection and validation of candidate reference genes for quantitative real-time PCR in Jasminum sambac Aiton[J]. Acta Agriculturae Boreali-Sinica, 35(06): 22-30.) [11] 任锐, 戴鹏辉, 李萌, 等. 2016. 珙桐实时定量PCR内参基因的筛选及稳定性评价[J]. 植物生理学报, 52(10): 1565-1575. (Ren R, Dai P H, Li M, et al.2016. Selection and stability evaluation of reference genes for real-time quantitative PCR in dove tree (Davidia involucrata Baill)[J]. Plant Physiology Journal, 52(10): 1565-1575.) [12] 盛德策, 李凤, 刘忠华. 2008. 植物体细胞胚发生的细胞生物学研究进展[J]. 西北植物学报, (01): 204-215. (Sheng D C, Li F, Liu Z H. 2008. Advances in the cytobiology of plant somatic embryogenesis[J]. Acta Botanica Boreali-Occidentalia Sinica, (01): 204-215.) [13] 王武, 李秀兰, 张献龙, 等. 1994. 油菜素内酯对陆地棉体细胞胚胎发生和根器官发生的影响[J]. 棉花学报, 6(02): 83-88. (Wang W, Li X L, Zhang X L, et al.1994. Effects of brassinolide on somatic embryogenesis and root organogenesis of Gossypium hirsutum Linn.[J]. Cotton Science, 6(02): 83-88.) [14] 徐娇. 2018. 棉花体细胞胚胎发生相关基因GhL1L1的克隆和功能验证[D]. 博士学位论文, 华中农业大学, 导师: 张献龙, pp. 74-77. (Xu J.2018. Cloning and functional verification of cotton somatic embryogenesis-related gene GhL1L1[D]. Thesis for Ph.D., Huazhong Agricultural University, Supervisor: Zhang X L, pp. 74-77.) [15] 于晓松, 王晓红, 李雪, 等. 2021. 钩藤实时荧光定量PCR分析中内参基因的筛选及稳定性评价[J]. 农业生物技术学报, 29(3): 587-597. (Yu X S, Wang X H, Li X, et al.2021. Screening and stability evaluation of reference genes in Uncaria rhynchophylla qRT-PCR analysis[J]. Journal of Agricultural Biotechnology, 29(3): 587-597.) [16] 袁伟, 万红建, 杨悦俭. 2012. 植物实时荧光定量PCR内参基因的特点及选择[J]. 植物学报, 47(04): 427-436. (Yuan W, Wan H J, Yang Y J.2012. Characterization and selection of reference genes for real-time quantitative qRT-PCR of plants[J]. Chinese Bulletin of Botany, 47(04): 427-436.) [17] 周成城, 荣俊冬, 谢德金, 等. 2021. 福建柏实时荧光定量PCR内参基因的选择[J]. 林业科学研究, 34(01): 137-145. (Zhou C C, Rong J D, Xie D J, et al.2021. Selection of reference genes of Fokienia hodginsii (Dunn) Henry et Thomas for real-time fluorescence quantitative PCR[J]. Forest Research, 34(01): 137-145.) [18] Anzidei M A, Bennici H, Schiff S, et al.2000. Organogenesis and somatic embryogenesis in Foeniculum vulgare: Histological observations of developing embryogenic callus[J]. Plant Cell, Tissue and Organ Culture, 61(01): 69-79. [19] Andersen C. L.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. [20] Axtell M J.2013. Classification and comparison of small RNAs from plants[J]. Annual Review of Plant Biology, 64(1): 137-159. [21] Bustin S A, Benes V, Garson J A, et al.2009. The MIQE guidelines: Mnimum information for publication of quantitative real-time PCR experiments[J]. Clinical Chemistry, 55(4): 611-622. [22] Chang E, Shi S, Liu J, et al.2012. Selection of reference genes for quantitative gene expression studies in Platycladus Orientalis (Cupressaceae) using real-time PCR[J]. PLOS ONE, 7(3): e33278. [23] Chen J, Sun Y, Sun F, et al.2011. Tobacco plants ectopically expressing the ammopiptanthus mongolicus AmCBL1 gene display enhanced tolerance to multiple abiotic stresses[J]. Plant Growth Regulation, 63(3): 259-269. [24] Chen Y, Li X, Su L, et al.2018. Genome-wide identification and characterization of long non-coding RNAs involved in the early somatic embryogenesis in Dimocarpus longan Lour[J]. BMC Genomics, 19(1): 1-19. [25] Chen J C,Wang Y Z, Wang C, et al.2020. LncRNA functions as a new emerging epigenetic factor in determining the fate of stem cells[J]. Frontiers in Genetics, 11: 277. [26] Cruz F, Kalaoun S, Nobile P, et al.2009. Evaluation of coffee reference genes for relative expression studies by quantitative real-time qRT-PCR[J]. Molecular Breeding, 23(4): 607-616. [27] Czechowski T, Stitt M, Altmann T, et al.2005. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis[J]. Plant Physiology, 139(1): 5-17. [28] Faccioli P, Ciceri G P, Provero P, et al.2007. A combined strategy of "in silico" transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies[J]. Plant Molecular Biology, 63(5): 679-688. [29] Galeano E, Vasconcelos T S, Ramiro D A, et al.2014. Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.)[J]. BMC Research Notes, 7: 464. [30] Kulcheski F R, Marcelino-Guimaraes F C, Nepomuceno A L, et al.2010. The use of microRNAs as reference genes for quantitative polymerase chain reaction in soybean[J]. Analytical Biochemistry, 406(2): 185-192. [31] Miao M, Yang X, Han X, et al.2011. Sugar signalling is involved in the sex expression response of monoecious cucumber to low temperature[J]. Journal of Experimental Botany, 62(2): 797-804. [32] Parra-Lobato M C, Gomez-Jimenez M C.2011. Polyamine-induced modulation of genes involved in ethylene biosynthesis and signalling pathways and nitric oxide production during olive mature fruit abscission[J]. Journal of Experimental Botany, 62(13): 4447-4465. [33] 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 Letters, 26(6): 509-515. [34] Postollec F, Falentin H, Pavan S, et al.2011. Recent advances in quantitative PCR (qRT-PCR) applications in food microbiology[J]. Food Microbiology, 28(5): 848-861. [35] Qiao M, Zhao Z, Song Y, et al.2012. Proper regeneration from in vitro cultured Arabidopsis thaliana requires the microRNA-directed action of an auxin response factor.[J].The Plant Journal, 71(1): 14-22. [36] Schaffer A A, Aravind L, Madden T L, et al.2001. Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements[J]. Nucleic Acids Research, 29(14): 2994-3005. [37] Silver N, Best S, Jiang J, et al.2006. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR[J]. BMC Molecular Biology, 7(1): 1-9. [38] Song S, Qi T, Huang H, et al.2011.The Jasmonate-ZIM domain proteins interact with the R2R3-MYB transcription factors MYB21 and MYB24 to affect jasmonate-regulated stamen development in Arabidopsis[J]. Plant Cell, 23(3): 1000-1013. [39] Taylor S, Wakem M, Dijkman G, et al.2010. A practical approach to RT-qPCR-Publishing data that conform to the MIQE guidelines[J]. Methods, 50(4): S1-S5. [40] Vanguilder H D, Vrana K E, Freeman W M.2008. Twenty-five years of quantitative qRT-PCR for gene expression analysis[J]. Biotechniques, 44(5): 619-626. [41] Vandesompele J, De Preter K, Pattyn F, et al.2002. Accurate normalization of real-time quantitative qRT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biology, 3(7): 1-11. [42] Voinnet O.2009. Origin, biogenesis, and activity of plant microRNAs[J]. Cell, 136(4): 669-687. [43] Varshney R, Budoff M J.2016. Garlic and heart disease[J]. Journal of Nutrition, 146(2): 416S-421S. [44] Yu D, Ma M, Liu Z, et al.2020. MOF-encapsulated nanozyme enhanced siRNA combo: Control neural stem cell differentiation and ameliorate cognitive impairments in Alzheimer's disease model[J]. Biomaterials, 255: 120160. [45] Zhou L, Wang Y, Peng Z.2011. Molecular characterization and expression analysis of chalcone synthase gene during flower development in tree peony (Paeonia suffruticosa)[J]. African Journal of Biotechnology, 10(8): 1275-1284. [46] Zanca A S, Vicentini R, Ortiz-Morea F A, et al.2010. Identification and expression analysis of microRNAs and targets in the biofuel crop sugarcane[J]. BMC Plant Biology, 10(1): 260.
