盐肤木APETALA3/DEFICIENS同源基因的克隆与功能分析

doi:10.11983/CBB24015

摘要/Abstract

摘要： APETALA3/DEFICIENS (AP3/DEF)基因为MADS-BOX基因家族的B类基因, 在花发育过程中主要参与调控花瓣和雄蕊的发育。对盐肤木AP3/DEF同源基因进行克隆及分析, 有助于探究其在盐肤木雄蕊发育过程中的作用。采用RT-PCR技术获得盐肤木AP3/DEF同源基因CDS; 利用NCBI CD Search对其序列和结构域进行比对分析; 利用酵母双杂交系统, 对AP3/DEF同源蛋白与盐肤木中其他MADS-box转录因子进行蛋白互作验证; 实时荧光PCR分析盐肤木AP3/DEF同源基因时空表达模式; 过表达拟南芥验证盐肤木AP3/DEF同源基因在花器官发育中的功能。本研究克隆得到2个盐肤木AP3/DEF同源基因, 分别命名为RcAP3 (GenBank: OR962160)和RcTM6 (GenBank: OR962159), 根据其氨基酸保守结构域比对及系统进化树分析, 发现这2个蛋白序列与漆树科的芒果和阿月浑子AP3/DEF同源基因编码蛋白亲缘关系最近。酵母双杂交结果表明RcAP3和RcTM6与盐肤木B类蛋白RcPI、C类蛋白RcAG和Rcag均存在互作关系, 但与A类和E类蛋白之间不存在互作关系。基因表达分析结果显示, 不同性别盐肤木花芽中RcAP3和RcTM6基因在花芽快速发育期高表达, 在花芽发育早期和开花后表达量较低; RcAP3在雌花、雄花和两性花花芽分化过程中均维持较高表达水平, 而RcTM6在两性花中显著表达, 在雄花和雌花中表达量很低。两性花快速生长期, RcAP3在花瓣和雄蕊中高表达且差异不大, 而RcTM6在雄蕊中的表达显著高于其他花器官。RcAP3基因能够恢复ap3-3突变体拟南芥花瓣和雄蕊表型, RcTM6过表达则会导致拟南芥花瓣、雄蕊和子房缩短, 花药败育。盐肤木中同属于AP3/DEF亚家族同源基因RcAP3和RcTM6存在功能分化。RcAP3具有促进花瓣和雄蕊发育的作用, 而RcTM6抑制雄蕊的发育。研究结果为进一步研究盐肤木性别分化的分子机制奠定基础。

关键词: 盐肤木, RcAP3, RcTM6, 表达模式, 功能分析

Abstract: The APETALA3/DEFICIENS (AP3/DEF) gene is a B-class gene of the MADS-box gene family, which is mainly involved in the regulation of petal and stamen development during flower development. Cloning and analysis of the AP3/DEF homologous genes from Rhus chinensis can help explore their role in the development of stamen. The CDS of AP3/DEF homologous gene was obtained using RT-PCR; its sequence and structural domains were compared and analyzed by NCBI CD Search; interactions between AP3/DEF homologous proteins and other MADS-box transcription factors in Rhus chinensis were verified by using yeast two-hybrid system; Real-time fluorescence PCR was used to analyze the spatial and temporal expression patterns of the AP3/DEF homologous genes; Overexpression in Arabidopsis was conducted to verify the function of the AP3/DEF homologous genes in flower organ development. Two coding sequences of AP3/DEF homologous genes in Rhus chinensis were cloned and named RcAP3 (GenBank: OR962160) and RcTM6 (GenBank: OR962159). Based on the alignment of their conserved amino acid domains and phylogenetic analysis, these two protein sequences showed the closest genetic relationship to the AP3/DEF homologous gene-encoded proteins in the Anacardiaceae family, including Mangifera indica and Pistacia vera. The yeast two-hybrid results showed that RcAP3 and RcTM6 interacted with the B-class protein RcPI, the C-class proteins RcAG and Rcag in Rhus chinensis, but not with the A-class and E-class proteins. Real-time fluorescence quantification results demonstrated that RcAP3 and RcTM6 genes were highly expressed during the rapid growth stage of flower buds in different sexes of Rhus chinensis, with lower expression levels during the early bud development and after flowering. RcAP3 gene exhibited high expression levels during buds differentiation in female, male, and hermaphrodite flowers, while RcTM6 showed significant expression in hermaphrodite flowers and very low expression in male and female flowers. During the rapid growth stage of hermaphrodite flowers, RcAP3 was highly expressed in both petals and stamens with minimal differences, whereas RcTM6 showed significantly higher expression in stamens compared to other floral organs. RcAP3 could restore the petal and stamen phenotypes in the ap3-3 mutant of Arabidopsis, while overexpression of RcTM6 resulted in shortened petals, stamens, and aborted ovaries in Arabidopsis. Functional differentiation exists between the AP3/DEF subfamily homologous genes, RcAP3 and RcTM6. RcAP3 promotes petal and stamen development, while RcTM6 inhibits stamen development. These findings provide a foundation for further research on the molecular mechanisms underlying sex differentiation in Rhus chinensis.

Key words: Rhus chinesnsis, RcAP3, RcTM6, expression pattern, functional analysis

顾磊, 张棋, 张霞, 杨冰冰, 王芳岚, 刘文, 陈发菊. 盐肤木APETALA3/DEFICIENS同源基因的克隆与功能分析. 植物学报, DOI: 10.11983/CBB24015.

Gu Lei, Zhang Qi, Zhang Xia, Yang Bingbing, Wang Fanglan, Liu Wen, Chen Faju. Cloning and Functional Analysis of APETALA3/DEFICIENS homologous gene from Rhus chinensis. Chinese Bulletin of Botany, DOI: 10.11983/CBB24015.

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参考文献

[1]景丹龙，夏燕，张守攻，王军辉(2016).黄金树B类MADS-box基因表达特征分析.植物学报, 51:210-217.
[2]李斌, 高洁莹, 龚力民，刘平安李顺祥 (2015)(2015).盐肤木果粕化学成分研究.中药材, 38:1209-1211.
[3]Broholm SK, P?ll?nen E, Ruokolainen S, T?htiharju S, Kotilainen M, Albert VA, Elomaa P, Teeri TH(2010).Functional characterization of B class MADS-box transcription factors in Gerbera hybrida.J Exp Bot, 61:75-85.
[4]Causier B, Castillo R, Xue Y, Schwarz-Sommer Z,Davies B(2010).Tracing the evolution of the floral homeotic B- and C-function genes through genome synteny.Mol Biol Evol, 27:2651-2664.
[5]Cao Xue, Liu Xiaoyan, Wang Xiaotian, Yang Mengxia, Giang Tongvan, Wang Jing, Liu Xiaolin, Sun Shuai, Wei Kai, Wang Xiaoxuan, Gao Jianchang, Du Yongchen, Qin Yong, Guo Yanmei, Huang Zejun(2019).B-class MADS-box TM6 is a candidate gene for tomato male sterile-1526.Theor Appl Genet, 132:2125-2135.
[6]Clough S.J., Bent A.F(1998).Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.The Plant J, 16:735-743.
[7]Doyle JJ(1994).Evolution of a plant homeotic multigene family: toward connecting molecular systematic and molecular developmental genetics.Syst Biol, 43:307-328.
[8]Fang Zhengwu, Qi Rui, Li Xiaofang, Liu Zhixiong(2014).Ectopic expression of FaesAP3,a Fagopyrum esculentum (Polygonaceae) AP3 orthologous gene rescues stamen development in an Arabidopsis ap3 mutant.Gene, 550:200-206.
[9]Goto K, Meyerowitz EM(1994).Function and regulation of the Arabidopsis floral homeotic gene PISTILLATA.Genes and Development, 8:1548-1560.
[10]Hernandez-Hernandez T, Martinez-Castilla LP, Alvarez-Buylla ER (2007).Functional diversification of B MADS-box homeotic regulators of flower development: adaptive evolution in protein–protein interaction domains after major gene duplication events.Mol Biol Evol, 24:465-481.
[11]Jack T, Brockman LL, Meyerowitz EM(1992).The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens.Cell, 68:683-697.
[12]Jack T(2001).Plant development going MADS.Plant Mol. Biol, 46:515-520.
[13]Jing Danlong, Chen Weiwei, Shi Min, Wang Dan, Xia Yan, Qiao He, Dang Jiangbo, Guo Qigao, Liang Guolu(2020).Ectopic expression of an Eriobotrya japonica APETALA3 ortholog rescues the petal and stamen identities in Arabidopsis ap3-3 mutant.Biochemical and Biophysical Research Communications, 523:33-38.
[14]Kenji Beppu, Hidemi Sumida, Ikuo Kataoka(2015).Cloning and characterisation of APETALA3 -like and PISTILLATA -like B class MADS-box genes from sweet cherry.Journal of Applied Horticulture, 17:87-91.
[15]Kim S, Yoo MJ, Albert VA, Farris JS, Soltis PS, Soltis DE(2004).Phylogeny and diversifi cation of B-function MADS-box genes in angiosperms: evolutionary and functional implications of a 260-millionyear-old duplication.Am J Bot, 91:2102-2118.
[16]Kramer EM, Irish VF(2000).Evolution of the petal and stamen developmental programs: evidence from comparative studies of the lower eudicots and basal angiosperms.International Journal of Plant Sciences, 161:S29-S40.
[17]Krasimir Rusanov, Natasha Kovacheva, Mila Rusanova, Marcus Linde, Thomas Debener & Ivan Atanassov(2019).Genetic control of flower petal number in Rosa x Damascena Mill ftrigintipetala.Biotechnology & Biotechnological Equipment, 33:597-604.
[18]Lai Xuelei, Hussein Daher, Antonin Galien, Veronique Hugouvieux, Chloe Zubieta(2019).Structural Basis for Plant MADS Transcription Factor Oligomerization.Comput Struct Biotechnol J, 17:946-53.
[19]Lamb RS, Irish VF(2003).Functional divergence within the APETALA3/PISTILLATA floral homeotic gene lineages .Proceedings of the National Academy of Sciences, 100:6558-6563.
[20]Li XiaoFang, Xu Jing, Yang Rang, Jia LinYan, Deng XinJie, Xiong LiJun, Zhang Xueping, Fang Qi, Zhang Wei, SunYue, Xu Ling(2013).Analysis of B-Class Genes NAP3L3 and NAP3L4 in Narcissus tazetta varchinensis.Plant Molecular Biology Reporter, 31:255-263.
[21]Li Meichen, Wang Andong, Zhang Yunqiang, Han Tingting, Guan Lu, Fan Dongxue, Liu Jiangyu, Xu Yongnan(2022).A comprehensive review on ethnobotanical,phytochemical and pharmacological aspects of Rhus chinensis Mill.Journal of Ethnopharmacology, 293:115288-115288.
[22]Litt A, Kramer EM(2010).The ABC model and the diversification of floral organ identity.Seminars in Cell & Devvelopment Biology, 21:129-137.
[23]Ma Nan, Cai Shengbao, Sun Yilin, Chu Chuanqi(2024).Chinese Sumac (Rhus chinensis Mill.) Fruits Prevent Hyperuricemia and Uric Acid Nephropathy in Mice Fed a High-Purine Yeast Diet.Nutrients, 16:184-184.
[24]Poupin MJ, Federici F, Medina C, Matus JT, Timmermann T, Arce-Johnson P(2007).Isolation of the three grape sub-lineages of B-class MADS-box TM6,PISTILLATA and APETALA3 genes which are differentially expressed during flower and fruit development.Gene, 404:10-24.
[25]Shen Gangxu, Yang Chihui, Shen ChiYen, Huang Kengshiang(2019).Origination and selection of ABCDE and AGL6 subfamily MADS-box genes in gymnosperms and angiosperms.Biologicahl Research, 52:1-15.
[26]Shen Gangxu, Jia Yong, Wang WeiLung(2021).Evolutionary divergence of motifs in B-class MADS-box proteins of seed plants.J of Biol Res-Thessaloniki, 28:1-10.
[27]Sommer H, Beltran JP, Huijser P, Pape H, L?nnig WE, Saedler H, Schwarz-Sommer Z(1990).Deficiens,a homeotic gene involved in the control of flower morphogenesis in Antirrhinum majus: the protein shows homology to transcription factors.The EMBO Journal, 9:605-613.
[28]Ye Ai, Zhang Chunling, Sun Yalin, Wang Wwining, He Yanghong, Bao Manzhu (2017)(2017).Characterization and Functional Analysis of Five MADS-Box B Class Genes Related to Floral Organ Identification in Tagetes erecta.PloS one, 12:e0169777-e0169777.
[29]Zhang Qiong, Wang Beiguo, Duan Ke, Wang Ligang, Wang Meng, Tang Xueming, Pan Aihu, Sui Shunzhao, Wang Guangdang(2011).The paleoAP3-type gene CpAP3,an ancestral B-class gene from the basal angiosperm Chimonanthus praecox,can affect stamen and petal development in higher eudicots.Development Genes and Evolution, 221:83-93.
[30]Zhang Haiqi, Han Wei, Linghu Tian, Zhao Zhixia, Wang Azheng, Zhai Rui, Yang Chengquan, Xu Lingfei, Wang Zhigang (2023).Overexpression of a pear B-class MADS-box gene in tomato causes male sterility.Fruit Research, 3:1-11.