School of Landscape and Architecture/Zhejiang Key Laboratory of Garden Plant Germplasm Innovation and Utilization/ Key Laboratory of Southern Garden Plant Germplasm Innovation and Utilization, State Forestry and Grassland Administration, Zhejiang A&F University, Hangzhou 311300, China
Abstract：Aquaporin (AQP) is a small molecule membrane intrinsic protein, which mediates the rapid and passive water transport and participates in transmembrane transport of plants. In this study, 41 genes of AQP gene family were identified according to non-redundant protein sequence database (Nr) annotation from the whole genome of Osmanthus fragrans. According to the analysis of amino acid sequence and phylogenetic tree, they were classified into 4 subgroups: 16 members of plasma membrane intrinsic proteins (PIP), 14 members of tonoplast intrinsic proteins (TIP), 8 members of nodulin-26-like intrinsic proteins (NIP), 3 members of small intrinsic proteins (SIP). The gene structure and conserved motif of amino acids in the same subgroup were highly conserved. The length of the AQP proteins ranged from 145 to 432 aa with molecular mass of 14.81 to 45.90 kD and the isoelectric points were from 4.84 to 9.81. The analysis of the expression patterns of OfPIPs and OfTIPs in different tissues of O. fragrans using qPCR showed that OfPIP1, OfPIP5, OfPIP12, OfPIP13, OfPIP14, OfPIP15, OfPIP16, OfTIP1, OfTIP3, OfTIP4, OfTIP6, OfTIP9 and OfTIP13 were specific expressed in flowers. Further study on their expression patterns during flower opening period of O. fragrans showed that, OfTIP6 was highly transcribed in the early flower opening period (S1~S2) of O. fragrans. It is speculated that OfTIP6 plays an important role in vacuole expansion at the early flower opening stage. However, the relative expression levels of OfPIP1, OfPIP13, OfPIP14, OfPIP15, OfPIP16 and OfTIP13 were significantly up-regulated at the late flower opening period (S4~S5) of O. fragrans (P<0.05). It is speculated that they are the key genes to exercise function at the late flower opening stage. This research provides a reference for further analyzing the biological function of OfAQPs protein.
[1] 付建新, 张超, 王艺光, 等. 2016. 桂花组织基因表达中荧光定量 PCR 内参基因的筛选[J]. 浙江农林大学学报, 33(5): 727-733. (Fu J X, Zhang C, Wang Y G, et al. 2016. Reference gene selection for quantitative real-time polymerase chain reaction (qRT-PCR) normalization in the gene expression of sweet Osmanthus tissues[J]. Journal of Zhejiang A ＆ F University, 33(5): 727-733.) [2] 李建军, 连笑雅, 王兰. 2019. 忍冬花蕾延迟开花与内源激素调控研究[J]. 园艺学报, 46(7): 1399-1408. (Li J J, Lian X Y, Wang L. 2019. Study on the regulation mechanism of endogenous hormones in delayed flowering of Lonicera japonica[J]. Acta Horticulturae Sinica, 46(7): 1399-1408.) [3] 李巧峡, 张丽, 王玉, 等. 2019. 赤霉素调控植物开花及花器官发育的研究进展[J]. 中国细胞生物学学报, 41(4): 746-758. (Li Q X, Zhang L, Wang Y, et al. 2019. The Research progress of gibberellin on the regulation of flowering and floral organ development in plant[J]. Chinese Journal of Cell Biology, 41(4): 746-758.) [4] 缪云锋, 周丹, 董彬,等. 2021. 桂花 OfNAC 转录因子鉴定及在花开放阶段的表达分析[J]. 浙江农林大学学报, 38(3): 433-444. (Miao Y F, Zhou D, Dong B, et al. 2021. Identification and expression analysis of OfNAC transcription factors in Osmanthus fragrans during flower opening stage[J]. Journal of Zhejiang A ＆ F University, 38(3): 433-444.) [5] 王英, 张超, 付建新,等. 2016. 桂花花芽分化和花开放研究进展[J]. 浙江农林大学学报, 33(2): 340-347. (Wang Y, Zhang C, Fu J X, et al. 2016. Progresses on flower bud differentiation and flower opening in Osmanthus fragrans[J]. Journal of Zhejiang A ＆ F University, 33(2): 340-347.) [6] 吴向阳,程朝泽,吕高强, 等. 2016. 芝麻 AQP 家族的全基因组序列鉴定及其特征分析[J]. 中国农业科学, 49(10): 1844-1858. (Wu X Y, Cheng C Z, Lu G Q, et al. 2016. Identification and characterization of the AQP gene family in sesame[J], Scientia Agricultura Sinica, 49(10): 1844-1858.) [7] 吴雪, 杜长霞, 杨冰冰, 等. 2015. 植物水通道蛋白研究综述[J]. 浙江农林大学学报, 32(5): 789-796. (Wu X, Du C X, Yang B B, et al. 2015. Research progress in plant aquaporins[J]. Journal of Zhejiang A ＆ F University, 32(5): 789-796.) [8] 徐德, 徐建俊, 李彪, 等. 2019. 植物水通道蛋白研究进展[J]. 分子植物育种, 17(14): 4674-4678. (Xu D, Xu J J, Li B, et al. 2019. Research advances for plant aquaporins[J]. Molecular Plant Breeding, 17(14): 4674-4678.) [9] 徐平珍, 刘涛, 杨莹, 等. 2007. 脱落酸在植物花发育过程中的作用[J]. 云南植物研究, 29(2): 215-222. (Xu P Z, Liu T, Y Y, et al. 2007. The role of abscisic acid in plant flowering[J]. Acta Botanica Yunnanica. 2007, 29(2): 215-222.) [10] Azad A K, Sawa Y, Ishikawa T, et al. 2004. Phosphorylation of plasma membrane aquaporin regulates temperature- dependent opening of tulip petals[J]. Plant and Cell Physiology, 45(5): 608-617. [11] Bienert G P, Chaumont F. 2010. Plant Aquaporins: Roles in water homeostasis, nutrition, and signaling processes[J]. Transporters and Pumps in Plant Signaling, 14369(4): 3-36. [12] Bots M, Vergeldt F, Wolters-Arts M, et al. 2005. Aquaporins of the PIP2 class are required for efficient anther dehiscence in tobacco[J]. Plant Physiology, 137(3): 1049-1056. [13] Chao J, Li Z, Sun Y, et al. 2021. MG2C: A user-friendly online tool for drawing genetic maps[J]. Molecular Horticulture, 1: 16. [14] Chaumont F, Barrieu F, Wojcik E, et al. 2001. Aquaporins constitute a large and highly divergent protein family in maize[J]. Plant Physiology, 125(3): 1206-1215. [15] Chen C, Chen H, Zhang Y, et al. 2020. TBtools: An integrative toolkit developed for interactive analyses of big biological data[J]. Molecular Plant, 13(8): 1194-1202. [16] Diehn T A, Pommerrenig B, Bernhardt N, et al. 2015. Genome-wide identification of aquaporin encoding genes in Brassica oleracea and their phylogenetic sequence comparison to Brassica crops and Arabidopsis[J]. Frontiers in Plant Science, 6: 166. [17] Federico A, Iker I, Rafael Z. 2014. Diversity and evolution of membrane intrinsic proteins[J], Biochimica et Biophysica Acta, 1840(5): 1468-1481. [18] Fu J X, Zhang C, Liu Y, et al. 2020. Transcriptomic analysis of flower opening response to relatively low temperatures in Osmanthus fragrans[J]. BMC Plant Biology, 20(1): 1-16. [19] Fukuhara T, Kirch H H, Bohnert H J, et al. 1999. Expression of Vp1 and water channel proteins during seed germination plant[J]. Plant, Cell and Environment, 22(4): 417-424. [20] Hohmann I, Bill R M, Kayingo I, et al. 2000. Microbial MIP channels[J]. Trends in Microbiology, 8(1): 33-38. [21] Irish V F. 2008. The Arabidopsis petal: A model for plant organogenesis[J].Trends in Plant Science, 13(8): 430-436. [22] Johnson K D, Höfte H, Chrispeels M J, et al. 1999. An intrinsic tonoplast protein of protein storage vacuoles in seeds is structurally related to a bacterial solute transporter (GIpF)[J]. Plant Cell, 2(6): 525-532. [23] Johanson U, Karlsson M, Johansson I, et al. 2001. The complete set of genes encoding major intrinsic proteins in Arabidopsis provides a framework for a new nomenclature for major intrinsic proteins in plants[J]. Plant Physiology, 126(4): 1358-1369. [24] Kalyaanamoorthy S, Minh B Q,Wong T K F, et al. 2017. ModelFinder: Fast model selection for accurate phylogenetic estimates[J]. Nature Methods, 14(6): 587-589. [25] Ma N, Xue J, Li Y, et al. 2008. Rh-PIP2; 1, a rose aquaporin gene, is involved in ethylene-regulated petal expansion[J]. Plant Physiology, 148(2): 894-907. [26] Nemoto K, Niiane T, Goto F, et al. 2022. Calcium-dependent protein kinase 16 phosphorylates and activates the aquaporin PIP2; 2 to regulate reversible flower opening in Gentiana scabra[J]. Plant Cell, 34(7): 2652-2670. [27] Nguyen L T, Schmidt H A, von Haeseler A, et al. 2015. IQ- TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[J]. Molecular Biology and Evolution, 32(1): 268-274. [28] Nystrom S L, Mckay D J. 2021. Memes: A motif analysis environment in R using tools from the MEME Suite[J]. PLOS Computational Biology, 17(9): e1008991-e1009004. [29] Panu A, Manohar J, Konstantin A, et al. 2012. ExPASy: SIB bioinformatics resource portal[J]. Nucleic Acids Research, 40: W597-W603. [30] Park W, Scheffler B E, Bauer P J, et al. 2010. Identification of the family of aquaporin genes and their expression in upland cotton (Gossypium hirsutum L.)[J]. BMC Plant Biology, 10: 142-17. [31] Preston G, Carroll T, Guggino W, et al. 1992. Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein[J]. Science, 256(5055): 385-392. [32] Reuscher S, Akiyama M, Mori C, et al. 2013. Genome-wide identification and expression analysis of aquaporins in tomato[J]. PLOS ONE, 8(11): e79052. [33] Sakurai J, Ishikawa F, Yamaguchi T, et al. 2005, Identification of 33 rice aquaporin genes and analysis of their expression and function[J]. Plant & Cell Physiology, 46(9): 1568-1577. [34] Tao P, Zhong X, Li B, et al. 2014. Genome-wide identification and characterization of aquaporin genes (AQPs) in Chinese cabbage (Brassica rapa ssp. pekinensis)[J]. Molecular Genetics and Genomics, 289(6): 1131-1145. [35] Uehlein N, Lovisolo C, Siefritz F, et al. 2003. The tobacco aquaporin NtAQP1 is a membrane CO2 pore with physiological functions[J]. Nature, 425(6959): 734-737. [36] Uehlein N, Otto B, Hanson D T, et al. 2008. Function of Nicotiana tabacum aquaporins as chloroplast gas pores challenges the concept of membrane CO2 permeability[J]. The Plant cell, 20(3): 648-657. [37] van Doorn W G, Kamdee C. 2014. Flower opening and closure: An update[J]. Journal of Experimental Botany, 65(20): 5749-5757. [38] Walz T, Hirai T, Murata K, et al. 1997. The three-dimensional structure of aquaporin-1[J]. Nature, 387(6633): 624-627. [39] Wouter G D, Uulke V M.2003, Flower opening and closure: A review[J]. Journal of Experimental Botany, 54(389): 1801-1812. [40] Xue J Q, Yang F, Gao J P. 2009. Isolation of Rh-TIP1; 1, an aquaporin gene and its expression in rose flowers in response to ethylene and water deficit[J]. Postharvest Biology and Technology, 51(3): 407-413. [41] Zhang D Y, Ali Z, Wang C B, et al. 2013. Genome-wide sequence characterization and expression analysis of major intrinsic proteins in soybean (Glycine max L.)[J]. PLOS ONE, 9(1): e56312. [42] Zhang S, Feng M, Chen W, et al. 2019. In rose, transcription factor PTM balances growth and drought survival via PIP2;1 aquaporin[J]. Nature Plants. 5(3): 290-299.
