Cloning and Bioinformatics Analysis of ZaAG Gene Related to Floral Organ Differentiation in Zanthoxylum armatum
WANG Yu, WANG Kai, LIU Rui, HE Yu-Xin, LI Hong-Jiao, XU Meng, TANG Zi-Yuan, HUI Wen-Kai*
College of Forestry/Sichuan Province Key Laboratory of Ecological Forestry Engineering on the Upper Reaches of the Yangtze River, Sichuan Agricultural University, Chengdu 611130, China
Abstract：Green prickly ash (Zanthoxylum armatum) dominated by parthenogenesis is shown to have a high proportion of male floral differentiation, which severely reduces the yield. But the underlying reason associated with stamen formation is not yet known. In previous studies, the floral organ differentiation is controlled by the ABC model in plants, and the C-class gene, named AGAMOUS (AG), plays a dominant role during the stamen and carpel differentiation processes. In this study, the coding sequence of ZaAG was isolated and identified based on our transcriptomic data and published genomic data profiles in Z. armatum, and the full CDS of ZaAG was cloned by PCR amplification technology. The results showed that the CDS length of ZaAG was 732 bp, encoding a total of 243 amino acids, including MADS domain composed of 77 amino acids and K-box domain composed of 86 amino acids. Phylogenetic analysis suggested that the AG protein was relatively conserved among the members of same family, and the ZaAG was closely related to the Rutaceae, such as Citrus clementina and C. sinensis. Additionally, a total of 148 transcription factor binding sites were predicted in ZaAG coding sequence, which were mainly involved in various reproductive growth and plant resistance pathways. The cis-elements analysis showed that the promoter region of ZaAG was enriched in the activation sites related to light signal, plant hormones, and stress. Furthermore, the ZaAG was not detected in the roots, stems, leaves, prickles and other vegetative organs, but the significantly high expression abundance was obtained during male and female flower differentiation process, especially the highest expression at the mature stage of male flowers (P<0.05), which indicated that ZaAG might be involved in the floral sex differentiation in Z. armatum. This study are of great significance for further analysis of the regulatory mechanisms related to male and female flower differentiation and breeding high-yielding germplasm in Z. armatum.
[1] 董燕梅, 张文颖, 凌正一, 等 . 2020. 转录因子调控植物萜类化合物生物合成研究进展[J]. 植物学报, 55(03): 340-350. (Dong Y M, Zhang W Y, Ling Z Y, et al.2020. Ad‐ vances in transcription factors regulating plant terpe‐ noids biosynthesis[J]. Chinese Bulletin of Botany, 55(03): 340-350.) [2] 位明明, 曾霞, 安泽伟, 等 . 2020. C 类花器官特征基因 AGA-MOUS (AG)调控植物花分生组织维持与终止研究进展[J]. 生物技术通报 , 36(01): 135-143. (Wei M M, Zeng X, An Z W, et al.2020. Advances in the maintenance and termination of floral meristem regulated by C-type floral organ gene AGAMOUS (AG)[J]. Biotechnology Bulletin, 36(01): 135-143.) [3] 文永, 李彪, 赵德刚, 等 . 2020. 杜仲糖基转移酶基因的启动子克隆及表达分析[J]. 农业生物技术学报 , 28(02): 223-231. (Wen Y, Li B, Zhao D G, et al.2020. Cloning and expression analysis of the promoter of glycosyl‐ transferase gene in Eucommia ulmoides[J]. Journal of Agricultural Biotechnology, 28(02): 223-231. [4] 张海霞 .2017. 藤椒花器结构及无融合生殖特性[D]. 硕士学位论文, 四川农业大学, 导师: 叶萌, pp. 21-23. (Zhang H X.2017. Flower structure and apomixis characteris‐ tics of Zanthoxylum armatum 'Tengjiao'[D]. Thesis for M. S., Sichuan Agricultural University, Supervisor: Ye M, pp. 21-23.) [5] Adal A M, Binson E, Remedios L, et al.2021. Expression of lavender AGAMOUS-like and SEPALLATA3-like genes promote early flowering and alter leaf morphology in Arabidopsis thaliana[J]. Planta, 254(3): 1-12. [6] An Y, Zhou Y Y, Han X, et al.2020. The GATA transcription factor GNC plays an important role in photosynthesis and growth in poplar[J]. Journal of Experimental Bota‐ ny, 71(6): 1969-1984. [7] Azuma M, Mitsuda N, Goto K, et al.2016. The petal-specific InMYB1 promoter functions by recognizing petaloid cells[J]. Plant and Cell Physiology, 57(3): 580-587. [8] Bao S J, Hua C M, Shen L S, et al.2020. New insights into gib‐berellin signaling in regulating flowering in Arabidopsis[J]. Journal of Integrative Plant Biology, 62(1): 118-131. [9] Bengoa Luoni S A, Cenci A, Moschen S, et al.2021. Genome-wide and comparative phylogenetic analysis of senes‐cence-associated NAC transcription factors in sunflower (Helianthus annuus)[J]. BMC Genomics, 22(1): 1-19. [10] Chen C J, Chen H, Zhang Y, et al.2020. TBtools: An integra‐tive toolkit developed for interactive analyses of big bio‐logical data[J]. Molecular Plant, 13(8): 1194-1202. [11] Cheng C Y, Krishnakumar V, Chan A P, et al.2017. Araport11: A complete reannotation of the Arabidopsis thaliana ref‐ erence genome[J]. The Plant Journal, 89(4): 789-804. [12] Coen E S, Meyerowitz E M.1991. The war of the whorls: Ge‐ netic interactions controlling flower development[J]. Na‐ ture, 353(6339): 31-37. [13] Ding F, Zhang S W, Chen H B, et al.2015. Promoter differ‐ence of LcFT1 is a leading cause of natural variation of flowering timing in different litchi cultivars (Litchi chi-nensis Sonn.)[J]. Plant Science, 241: 128-137. [14] Dong Z F, Zhao X L, Liu J J, et al.2022. Detection and simul‐ taneous differentiation of three co-infected viruses in Zanthoxylum armatum[J]. Plants, 11(9): 1242. [15] Dong H Q, Liu L X, Fan X Q, et al.2019. The artificial pro‐ moter rMdAG2I confers flower-specific activity in Malus [J]. International Journal of Molecular Science, 20(18):4551. [16] Duren Z N, Wang Y L, Wang J G, et al.2019. Hierarchical graphical model reveals HFR1 bridging circadian rhythm and flower development in Arabidopsis thaliana [J]. NPJ Systems Biology and Applications, 5(1): 1-11. [17] Fei X T, Shi Q Q, Qi Y C, et al.2021. ZbAGL11, a class D MADS-box transcription factor of Zanthoxylum bungea- num, is involved in sporophytic apomixis[J]. Horticul‐ ture Research, 8(1): 23. [18] Fei X T, Shi Q Q, Yang T X, et al.2018. Expression stabilities of ten candidate reference genes for RT-qPCR in Zan- thoxylum bungeanum Maxim[J]. Molecules, 23(4): 802. [19] Gao Y R, Sun J C, Sun Z L, et al.2020. The MADS-box tran‐ scription factor CmAGL11 modulates somatic embryo‐ genesis in Chinese chestnut (Castanea mollissima Blume)[J]. Journal of Integrative Agriculture, 19(4):1033-1043. [20] Gu C, Guo Z H, Hao P P, et al.2017. Multiple regulatory roles of AP2/ERF transcription factor in angiosperm[J]. Botanical Studies, 58(1): 1-8. [21] Guan H L, Wang H, Huang J J, et al.2021. Genome-wide identification and expression analysis of MADS-box family genes in litchi (Litchi chinensis Sonn.) and their involvement in floral sex determination[J]. Plants, 10(10): 2142. [22] Han P, García-Ponce B, Fonseca-Salazar G, et al.2008. AGA‐ MOUS-LIKE 17, a novel flowering promoter, acts in a FT-independent photoperiod pathway[J]. The Plant Jour‐ nal, 55(2): 253-265. [23] He X, Zhu L F, Wassan G M, et al.2018. GhJAZ2 attenuates cotton resistance to biotic stresses via the inhibition of the transcriptional activity of GhbHLH171[J]. Molecular Plant Pathology, 19(4): 896-908. [24] Heijmans K, Morel P, Vandenbussche M.2012. MADS-box genes and floral development: The dark side[J]. Journal of Experimental Botany, 63(15): 5397-5404. [25] Hu B, Jin J P, Guo A Y, et al.2015. GSDS 2.0: An upgraded gene feature visualization server[J]. Bioinformatics, 31(8): 1296-1297. [26] Hu R, Yuan C, Niu Y, et al.2020. Regulation of plant MYB transcription factors in anther development[J]. Chinese Journal of Biotechnology, 36(11): 2277-2286. [27] Hui W K, Wang J Y, Ma L X, et al.2021a. Identification of key genes in the biosynthesis pathways related to terpe‐noids, alkaloids and flavonoids in fruits of Zanthoxylum armatum[J]. Scientia Horticulturae, 290: 110523. [28] Hui W K, Liu M Q, Wu G J, et al.2021b. Ectopic expression of an AGAMOUS homologue gene in Jatropha curcas causes early flowering and heterostylous phenotypes[J]. Gene, 766: 145141. [29] Hui W K, Zhao F Y, Wang J Y, et al.2020. De novo transcrip‐ tome assembly for the five major organs of Zanthoxy- lum armatum and the identification of genes involved in terpenoid compound and fatty acid metabolism[J]. BMC Genomics, 21(1): 1-15. [30] Hui W K, Fan J T, Liu X Z, et al.2022. Integrated transcrip‐ tome and plant growth substance profiles to identify the regulatory factors involved in floral sex differentiation in Zanthoxylum armatum DC[J]. Frontiers in Plant Sci‐ ence, 13: 976338. [31] Ito T, Ng K H, Lim T S, et al.2007. The homeotic protein AG‐ AMOUS controls late stamen development by regulat‐ ing a jasmonate biosynthetic gene in Arabidopsis[J]. The Plant Cell, 19(11): 3516-3529. [32] Jagadish S V K, Way D A, Sharkey T D.2021. Plant heat stress: Concepts directing future research[J]. Plant, Cell & Environment, 44(7): 1992-2005. [33] Jibran R, Tahir J, Cooney J, et al.2017. Arabidopsis AGA‐ MOUS regulates sepal senescence by driving jasmonate production[J]. Frontiers in Plant Science, 8: 2101. [34] Jung H, Jo S H, Jung W Y, et al.2020. Gibberellin promotes bolting and flowering via the floral integrators RsFT and RsSOC1-1 under marginal vernalization in radish[J]. Plants, 9(5): 594. [35] Kalia N K, Singh B, Sood R P.1999. A new amide from Zan- thoxylum armatum[J]. Journal of Natural Products, 62(2): 311-312. [36] Kato T, Hibino T.2009. Isolation and expression analysis of AGAMOUS-like genes from Eucalyptus grandis[J]. Plant Biotechnology. 26(1): 121-124. [37] Klocko A L, Brunner A M, Ma C, et al.2020. RNAi of AGA‐ MOUS genes in sweetgum alters reproductive organ identity and decreases fruit persistence[J]. Plant Direct, 4(5): e00225. [38] Lescot M, Déhais P, Thijs G, et al.2002. PlantCARE, a data‐base of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J]. Nucleic Acids Research, 30(1): 325-327. [39] Li H W, Wang L Y, Mai Y N, et al.2021. Phytohormone and integrated mRNA and miRNA transcriptome analyses and differentiation of male between hermaphroditic flo‐ral buds of andromonoecious Diospyros kaki Thunb[J]. BMC Genomics, 22(1): 1-19. [40] Li H Y, Li L X, ShangGuan G D, et al.2020a. Genome-wide identification and expression analysis of bZIP gene fam‐ ily in Carthamus tinctorius L.[J]. Scientific Reports, 10(1): 1-15. [41] Li W X, Pang S Y, Lu Z G, et al.2020b. Function and mecha‐ nism of WRKY transcription factors in abiotic stress re‐ sponses of plants[J]. Plants, 9(11): 1515. [42] Liu X M, He X, Liu Z B, et al.2022. Transcriptome mining of genes in Zanthoxylum armatum revealed ZaMYB86 as a negative regulator of prickly development[J]. Genom‐ics, 114(3): 110374. [43] Liu H L, Wang X B, Wang G B, et al.2021a. The nearly com‐ plete genome of Ginkgo biloba illuminates gymnosperm evolution[J]. Nature Plants, 7(6): 748-756. [44] Liu Z X, Zhang D S, Zhang W W, et al.2021b. Molecular cloning and expression profile of class E genes related to sepal development in Nelumbo nucifera[J]. Plants, 10(8): 1629-1629. [45] Liu J H, Liu L, Li Y J, et al.2015. Role for the banana AGA‐ MOUS-like gene MaMADS7 in regulation of fruit ripen‐ ing and quality[J]. Physiologia Plantarum, 155(3): 217-231. [46] Liu D F, Shi S P, Hao Z J, et al.2019. OsbZIP81, A Homo‐ logue of Arabidopsis VIP1, may positively regulate JA levels by directly targetting the genes in JA signaling and metabolism pathway in rice[J]. International Journal of Molecular Sciences, 20(9): 2360. [47] Liu Y, Hao X Y, Lu Q H, et al.2020. Genome-wide identifica‐ tion and expression analysis of flowering-related genes reveal putative floral induction and differentiation mech‐ anisms in tea plant (Camellia sinensis)[J]. Genomics, 112(3): 2318-2326. [48] Long Y Q, Liu X, Zeng J, et al.2021. Cloning and expression of AGL19 gene in two Lonicera macranthoides varieties[J]. Journal of Genetics, 100(1): 1-10. [49] Maier A T, Stehling-Sun S, Wollmann H, et al.2009. Dual roles of the bZIP transcription factor PERIANTHIA in the control of floral architecture and homeotic gene ex‐ pression[J]. Development, 136(10): 1613-1620. [50] Monniaux M, Vandenbussche M.2018. How to evolve a peri‐anth: a review of cadastral mechanisms for perianth identity[J]. Frontiers in Plant Science, 9: 1573. [51] Müller N A, Kersten B, Leite Montalvão A P, et al.2020. A single gene underlies the dynamic evolution of poplar sex determination[J]. Nature Plants, 6(6): 630-637. [52] Okagu I U, Ndefo J C, Aham E C, et al.2021. Zanthoxylum Species: A comprehensive review of traditional uses, phytochemistry, pharmacological and nutraceutical ap‐ plications[J]. Molecules, 26(13): 4023. [53] Qu Y Q, Kong W L, Wang Q, et al.2021. Genome-wide iden‐ tification MIKC-type MADS-box gene family and their roles during development of floral buds in wheel wing‐nut (Cyclocarya paliurus)[J]. International Journal of Mo‐lecular Sciences, 22(18): 10128. [54] Su Y L, Liu J X, Liang W Q, et al.2019. Wheat AGAMOUS LIKE 6 transcription factors function in stamen develop‐ ment by regulating the expression of Ta APETALA3[J]. Development, 146(20): 177527. [55] Sun Y K, Fan Z Q, Li X L, et al.2014. Distinct double flower varieties in Camellia japonica exhibit both expansion and contraction of C-class gene expression[J]. BMC Plant Biology, 14(1): 1-11. [56] Theissen G, Melzer R, Rumpler F.2016. MADS-domain tran‐ scription factors and the floral quartet model of flower development: Linking plant development and evolution[J]. Development, 143(18): 3259-3271. [57] Tuskan G A, DiFazio S, Jansson S.et al.2006. The genome of black cottonwood, Populus trichocarpa (Torr. & Gray)[J]. Science, 313(5793): 1596-1604. [58] Uemura A, Yamaguchi N, Xu Y, et al.2018. Regulation of flo‐ ral meristem activity through the interaction of AGA‐ MOUS, SUPERMAN, and CLAVATA3 in Arabidopsis [J]. Plant Reproduction, 31(1): 89-105. [59] Waheed S, Peng Y, Zeng L H.2020. Identification and charac‐ terization of DlGI promoter involved in photoperiod, light intensity, hormone, and DlELF4 response from lon‐ gan[J]. Journal of the American Society for Horticultur‐ al Science, 145(6): 340-348. [60] Wang X, Song M Y, Flaishman M A, et al.2021a. AGA‐MOUS Gene as a new sex-identification marker in Fig (Ficus carica L.) is more efficient than RAN1[J]. Fron‐ tiers in Plant Science, 12: 755358. [61] Wang M C, Tong S F, Ma T, et al.2021b. Chromosome-level genome assembly of Sichuan pepper provides insights into apomixis, drought tolerance, and alkaloid biosyn‐ thesis[J]. Molecular Ecology Resources, 21(7): 2533-2545. [62] Wang Y P, Tang H B, DeBarry J D, et al.2012a. MCScanX: A toolkit for detection and evolutionary analysis of gene synteny and collinearity[J]. Nucleic Acids Research, 40(7): e49-e49. [63] Wang L Y, Li H W, Sun P, et al.2020. Effects of plant growth regulators, soil moisture contents, and carbon/nitrogen ratios on sex differentiation in persimmon (Diospyros ka-ki Thunb.) flowers[J]. Journal of Plant Growth Regula‐tion, 40(3): 1121-1138. [64] Wang Y, Zhang X M, Liu Z X, et al.2012b. Isolation and char‐ acterization of an AGAMOUS-like gene from Hosta plan- taginea[J]. Molecular Biology, 39: 2875-2881. [65] Wei D H, Liu M J, Chen H, et al.2018. INDUCER OF CBF EXPRESSION 1 is a male fertility regulator impacting anther dehydration in Arabidopsis[J]. PLOS Genetics, 14(10): e1007695. [66] Winter C M, Austin R S, Blanvillain-Baufumé S, et al.2011. LEAFY target genes reveal floral regulatory logic, cis motifs, and a link to biotic stimulus response[J]. Devel‐ opmental Cell, 20(4): 430-443. [67] Wu J, Liu Y, Tang L, et al.2011. A study on structural features in early flower development of Jatropha curcas L. and the classification of its inflorescences[J]. African Jour‐ nal of Agricultural Research, 6(2): 275-284. [68] Xu D, Zhuo Z, Wang R, et al.2019. Modeling the distribution of Zanthoxylum armatum in China with MaxEnt model‐ ing[J]. Global Ecology and Conservation, 19: e00691. [69] Xu Q, Chen L L, Ruan X A, et al.2013. The draft genome of sweet orange (Citrus sinensis)[J]. Nature Genetics, 45(1): 59-66. [70] Xue L J, Wu H T, Chen Y N, et al.2020. Evidences for a role of two Y-specific genes in sex determination in Populus deltoides[J]. Nature Communications, 11(1): 1-12. [71] Yang W L, Lou X Y, Li J, et al.2017a. Cloning and functional analysis of MADS-box genes, TaAG-A and TaAG-B, from a wheat K-type cytoplasmic male sterile line[J]. Frontiers in Plant Science, 8: 1081. [72] Yang Q, Chen Q J, Zhu Y D, et al.2017b. Identification of MdDof genes in apple and analysis of their response to biotic or abiotic stress[J]. Functional Plant Biology, 45(5): 528-541. [73] Yang Q, Dong B Y, Wang L T, et al.2020. CDPK6 phosphory‐ lates and stabilizes MYB30 to promote hyperoside bio‐ synthesis that prolongs the duration of full-blooming in okra[J]. Journal of Experimental Botany, 71(14): 4042-4056. [74] Ye Z L, Zang Q L, Cheng D X, et al.2022. Over-expression of larch DAL1 accelerates life-cycle progression in Ara-bidopsis[J]. Forests, 13(6): 953. [75] Yin X J, Zhang Y B, Zhang L, et al.2021. Regulation of MYB transcription factors of anthocyanin synthesis in lily flowers[J]. Frontiers in Plant Science, 12: 761668. [76] Yu L H, Wu J, Zhang Z S, et al.2017. Arabidopsis MADS-box transcription factor AGL21 acts as environmental sur‐ veillance of seed germination by regulating ABI5 ex‐ pression[J]. Molecular Plant, 10(6): 834-845. [77] Yu H, Ito T, Zhao Y X, et al.2004. Floral homeotic genes are targets of gibberellin signaling in flower development[J]. Proceedings of the National Academy of Sciences of the USA, 101(20): 7827-7832. [78] Zeng X C, Zhou X, Zhang W, et al.1999. Opening of rice flo‐ ret in rapid response to methyl jasmonate[J]. Journal of Plant Growth Regulation, 18(4): 153-158. [79] Zhang X, Tang N, Liu X M, et al.2020a. Comparative tran‐scriptome analysis identified differentially expressed genes between male and female flowers of Zanthoxylum armatum var. novemfolius[J]. Agronomy, 10(2): 283. [80] Zhang X D, Fatima M, Zhou P, et al.2020b. Analysis of MADS-box genes revealed modified flowering gene net‐ work and diurnal expression in pineapple[J]. BMC Ge‐ nomics, 21(1): 1-16. [81] Zhang C L, Wei L D, Wang W J, et al.2020c. Identification, characterization and functional analysis of AGAMOUS subfamily genes associated with floral organs and seed development in Marigold (Tagetes erecta)[J]. BMC Plant Biology, 20(1): 1-17. [82] Zhang J Y, Wang M, Mo Z H, et al.2016. Molecular charac‐ terization and functional analysis of an AGAMOUS-like gene CiAG from pecan[J]. HortScience, 51(6): 664-668. [83] Zhao Y, Liu R, Xu Y T, et al.2019. AGLF provides C-func‐ tion in floral organ identity through transcriptional regu‐ lation of AGAMOUS in Medicago truncatula[J]. Pro‐ ceedings of the National Academy of Sciences of the USA, 116(11): 5176-5181. [84] Zhou F Y, Yu Q, Zhang Y, et al.2021. Overexpression of AG‐ AMOUS-like gene PfAG5 promotes early flowering in Polypogon fugax[J]. Functional Plant Biology, 48(8): 793-801. [85] Zhou A, Sun H W, Dai S Y, et al.2019. Identification of tran‐ scription factors involved in the regulation of flowering in Adonis amurensis through combined RNA-seq tran‐ scriptomics and iTRAQ proteomics[J]. Genes, 10(4):305.
