‘鲁星’桃中10个MADS-box基因克隆和表达分析

doi:10.3864/j.issn.0578-1752.2016.23.012

摘要/Abstract

摘要： 【目的】克隆‘鲁星’桃（Prunus persica var. nectarina ‘Luxing’）中参与调控营养和生殖生长的MADS-box（PpMADS）基因，研究其在不同组织器官中的表达特性，为解析该基因在花发育和果实发育及成熟过程中的功能奠定基础。【方法】利用同源比对和RT-PCR技术，克隆获得‘鲁星’桃中10个PpMADS全长cDNA序列，并进行生物信息学相关分析；采用RT-PCR技术检测PpMADSs在茎、叶、萼片、子房、雄蕊、花瓣等组织以及花发育7个阶段和果实发育5个阶段的表达特性。【结果】测序结果显示，获得10个PpMADSs（PpMADS11、12、19、20、21、22、28、29、30和31；GenBank登录号分别为KU559577、KU559578、KU559585、KU559586、KU559587、KU559588、KU559594、KU559595、KU559596和KU559597），其开放阅读框（open reading frame，ORF）分别为522、279、1 065、828、723、600、636、534、750和480 bp。进化分析表明，PpMADS11属于AP3亚组，PpMADS12是AGL17亚组，PpMADS19属于MIKC*组，PpMADS20、21和22同被分为Mα组，PpMADS28、29、30和31同属于Mγ组。亚细胞定位预测结果显示，10个PpMADS蛋白均定位于细胞核中。启动子分析显示，PpMADSs启动子区域含有多个顺式作用元件，包括光响应元件、防御及逆境响应元件、干旱诱导的MYB结合位点、热激响应元件、低温响应元件、真菌效应子响应元件、伤害响应元件、厌氧响应元件、GA响应元件、Auxin响应元件、MeJA响应元件、ABA响应元件、SA响应元件和乙烯响应元件。半定量RT-PCR和qRT-PCR结果显示，PpMADS11在茎、叶、萼片、子房、雄蕊、花瓣、花发育和果实发育中表达；PpMADS12在茎、叶、萼片、子房、雄蕊、花瓣和花发育中表达；PpMADS19在萼片、雄蕊、花瓣和花发育中（苗期除外）表达；Mα组和Mγ组所有成员在茎、叶、萼片、子房、雄蕊、花瓣和花发育中均有表达，部分成员在果实发育中表达。【结论】10个PpMADS在‘鲁星’桃的营养生长以及花和果实发育过程中可能具有重要的调控作用。

关键词: '鲁星'桃, MADS-box, 转录因子, 基因克隆, 表达分析

Abstract: 【Objective】The aim of this study is to characterize the novel peach (Prunus persica var. nectarina ‘Luxing’) MADS-box genes (PpMADSs) involved in regulation of vegetative and reproductive growth. The transcriptional levels of PpMADSs in different tissues were determined to provide a basis for studying the related function of PpMADSs in flower development, fruit development and ripening.【Method】The full-length cDNA sequences of PpMADSs form ‘Luxing’ peach were isolated by homologous alignment and RT-PCR confirmation, the obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods; the expression levels of PpMADSs were detected in stem, leaf, sepal, ovary, stamen, petal, 7 stages of flower development and 5 stages of fruit development using RT-PCR.【Result】The sequencing results showed that ten cDNAs (designated as PpMADS11, 12, 19, 20, 21, 22, 28, 29, 30 and 31; GenBank accession No. KU559577, KU559578, KU559585, KU559586, KU559587, KU559588, KU559594, KU559595, KU559596 and KU559597) contained open reading frame (ORF) of 522, 279, 1 065, 828, 723, 600, 636, 534, 750 and 480 bp, respectively. The results of phylogenetic analysis revealed that PpMADS11, 12, and 19 belong to AP3, AGL17 and MIKC* subgroups, respectively; and PpMADS20, 21 and 22 belong to Mα group; PpMADS28, 29, 30 and 31 belong to Mγ group. The results of subcellular localization prediction showed that all PpMADS proteins were located in the nucleus. The results of promoter analysis indicated that there were multiple putative cis-acting elements involved in light responsiveness, defense and stress responsiveness, MYB binding site was involved in drought-inducibility, heat stress responsiveness, low-temperature responsiveness, fungal elicitor responsive element, wound-responsive element, anaerobic induction element, gibberellin-responsive element, auxin-responsive element, MeJA-responsiveness, abscisic acid responsiveness, salicylic acid responsiveness and ethylene-responsive element. Semi RT-PCR and qRT-PCR results showed that PpMADS11was expressed in stem, leaf, sepal, ovary, stamen, petal and during flower and fruit development. PpMADS12 was expressed in stem, leaf, sepal, ovary, stamen, petal and during flower development. PpMADS19 was expressed in sepal, stamen, petal and during flower development ( except bud stage). All members in Mα and Mγ groups were expressed in stem, leaf, sepal, ovary, stamen, petal and during flower development, some members were expressed during fruit development.【Conclusion】These results indicated that ten PpMADS genes have crucial regulatory roles in ‘Luxing’ peach vegetative growth, flower and fruit development processes.

Key words: ‘Luxing&rsquo, peach, MADS-box, transcription factor, gene cloning, expression analysis

李慧峰，贾厚振，董庆龙，冉昆，王宏伟. ‘鲁星’桃中10个MADS-box基因克隆和表达分析[J]. 中国农业科学, 2016, 49(23): 4593-4605.

LI Hui-feng, JIA Hou-zhen, DONG Qing-long, RAN Kun, WANG Hong-wei. Cloning and Expression Analysis of Ten MADS-box Genes in Peach (Prunus persica var. nectarina ‘Luxing’)[J]. Scientia Agricultura Sinica, 2016, 49(23): 4593-4605.

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参考文献

[1] RIECHMANN J L, MEYEROWITZ E M. MADS domain proteins in plant development. Biological Chemistry, 1997, 378(10): 1079-1101. [2] MESSENGUY F, DUBOIS E. Role of MADS box proteins and their cofactors in combinatorial control of gene expression and cell development. Gene, 2003, 316: 1-21. [3] 刘菊华, 徐碧玉, 张静, 金志强. MADS-box转录因子的相互作用及对果实发育和成熟的调控. 遗传, 2010, 32(9): 893-902. LIU J H, XU B Y, ZHANG J, JIN Z Q. The interaction of MADS-box transcription factors and manipulating fruit development and ripening. Hereditas, 2010, 32(9): 893-902. (in Chinese) [4] KAUFMANN K, MELZER R, THEISSEN G. MIKC-type MADS- domain proteins: structural modularity, protein interactions and network evolution in land plants. Gene, 2005, 347(2): 183-198. [5] HENSCHEL K, KOFUJI R, HASEBE M, SAEDLER H, MUNSTER T, THEISSEN G. Two ancient classes of MIKC-type MADS-box genes are present in the moss Physcomitrella patens. Molecular Biology and Evolution, 2002, 19(6): 801-814. [6] TIAN Y, DONG Q L, JI Z R, CHI F M, CONG P H, ZHOU Z S. Genome-wide identification and analysis of the MADS-box gene family in apple. Gene, 2015, 555(2): 277-290. [7] WEIGEL D, MEYEROWITZ E M. The ABCs of floral homeotic genes. Cell, 1994, 78(2): 203-209. [8] MA H, DEPAMPHILIS C. The ABCs of floral evolution. Cell, 2000, 101(1): 5-8. [9] IRELAND H S, YAO J L, TOMES S, SUTHERLAND P W, NIEUWENHUIZEN N, GUNASEELAN K, WINZ R A, DAVID K M, SCHAFFER R J. Apple SEPALLATA1/2-like genes control fruit flesh development and ripening. The Plant Journal, 2013, 73(6): 1044-1056. [10] FUJISAWA M, SHIMA Y, NAKAGAWA H, KITAGAWA M, KIMBARA J, NAKANO T, KASUMI T, ITO Y. Transcriptional regulation of fruit ripening by tomato FRUITFULL homologs and associated MADS box proteins. The Plant Cell, 2014, 26(1): 89-101. [11] FERRANDIZ C, LILJEGREN S J, YANOFSKY M F. Negative regulation of the SHATTERPROOF genes by FRUITFULL during Arabidopsis fruit development. Science, 2000, 289(5478): 436-438. [12] VREBALOV J, RUEZINSKY D, PADMANABHAN V, WHITE R, MEDRANO D, DRAKE R, SCHUCH W, GIOVANNONI J. A MADS-box gene necessary for fruit ripening at the tomato ripening- inhibitor (rin) locus. Science, 2002, 296(5566): 343-346. [13] ITO Y, KITAGAWA M, IHASHI N, YABE K, KIMBARA J, YASUDA J, ITO H, INAKUMA T, HIROI S, KASUMI T. DNA-binding specificity, transcriptional activation potential, and the rin mutation effect for the tomato fruit-ripening regulator RIN. The Plant Journal, 2008, 55(2): 212-223. [14] MARA C D, IRISH V F. Two GATA transcription factors are downstream effectors of floral homeotic gene action in Arabidopsis. Plant Physiology, 2008, 147(2): 707-718. [15] KAUFMANN K, MUIÑO J M, JAUREGUI R, AIROLDI C A, SMACZNIAK C, KRAJEWSKI P, ANGENENT G C. Target genes of the MADS transcription factor SEPALLATA3: Integration of developmental and hormonal pathways in the Arabidopsis flower. PLOS Biology, 2009, 7(4): 854-875. [16] WELLS C E, VENDRAMIN E, TARODO S J, VERDE I, BIELENBERG D G. A genome-wide analysis of MADS-box genes in peach [Prunus persica (L.) Batsch]. BMC Plant Biology, 2015, 15(1): 41. [17] 王传增, 余贤美, 董庆龙, 张安宁, 刘伟, 董飞, 王淑珍, 王长君. 桃已知 MADS-box转录因子的生物信息学及花发育表达分析. 核农学报, 2015, 29(5): 849-858. WANG C Z, YU X M, DONG Q L, ZHANG A N, LIU W, DONG F, WANG S Z, WANG C J. Bioinformatic and expression analysis on the known MADS-box transcription factors at different development stages of flower in peach. Journal of Nuclear Agricultural Sciences, 2015, 29(5): 849-858. (in Chinese) [18] SUI S, LUO J, MA J，ZHU Q, LEI X, LI M. Generation and analysis of expressed sequence tags from Chimonanthus praecox (Wintersweet) flowers for discovering stress-responsive and floral development- related genes. Comparative and Functional Genomics, 2012, doi:10. 1155/2012/134596. [19] 马婧, 孙文婷, 王晶, 眭顺照, 李名扬. 蜡梅胚胎晚期丰富蛋白基因CpLEA的克隆及表达分析. 园艺学报, 2014, 41(8): 1663-1672. MA J, SUN W T, WANG J, MU S Z, LI M Y. Cloning and expression analysis of a late embryogenesis abundant protein gene CpLEA from Chimonanthus praecox. Acta Horticulturae Sinica, 2014, 41(8): 1663-1672. (in Chinese) [20] 谷彦冰, 冀志蕊, 迟福梅, 乔壮, 徐成楠, 张俊祥, 董庆龙, 周宗山. 苹果WRKY基因家族生物信息学及表达分析. 中国农业科学, 2015, 48(16): 3221-3238. GU Y B, JI Z R, CHI F M, QIAO Z, XU C N, ZHANG J X, DONG Q L, ZHOU Z S. Bioinformatics and expression analysis of the WRKY gene family in apple. Scientia Agricultura Sinica, 2015, 48(16): 3221-3238. (in Chinese) [21] 谷彦冰, 冀志蕊, 迟福梅, 乔壮, 徐成楠, 张俊祥, 周宗山, 董庆龙. 桃WRKY基因家族全基因组鉴定和表达分析. 遗传, 2016, 38(3): 254-270. GU Y B, JI Z R, CHI F M, QIAO Z, XU C N, ZHANG J X, ZHOU Z S, DONG Q L. Genome-wide identification and expression analysis of the WRKY gene family in peach. Hereditas (Beijing), 2016, 38(3): 254-270. (in Chinese) [22] PAENICOVÁ L, DE-FOLTER S, KIEFFER M, HORNER D S, FAVALLI C, BUSSCHER J, COOK H E, INGRAM R M, KATER M M, DAVIES B, ANGENENT G C, COLOMBO L. Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: New openings to the MADS world. The Plant Cell, 2003, 15(7): 1538-1551. [23] ARORA R, AGARWAL P, RAY S, SINGH A K, SINGH V P, TYAGI A K, KAPOOR S. MADS-box gene family in rice: genome-wide identification, organization and expression profiling during reproductive development and stress. BMC Genomics, 2007, 8(1): 242. [24] VERDE I, ABBOTT A G, SCALABRIN S, JUNG S, SHU S, MARRONI F, ZHEBENTYAYEVA T, DETTORI M T, GRIMWOOD J, CATTONARO F, ZUCCOLO A, ROSSINI L, JENKINS J, VENDRAMIN E, MEISEL L A, DECROOCQ V, SOSINSKI B, PROCHNIK S, MITROS T, POLICRITI A, CIPRIANI G, DONDINI L, FICKLIN S, GOODSTEIN D M, XUAN P, DEL FABBRO C, ARAMINI V, COPETTI D, GONZALEZ S, HORNER D S, FALCHI R, LUCAS S, MICA E, MALDONADO J, LAZZARI B, BIELENBERG D, PIRONA R, MICULAN M, BARAKAT A, TESTOLIN R, STELLA A, TARTARINI S, TONUTTI P, ARÚS P, ORELLANA A, WELLS C, MAIN D, VIZZOTTO G, SILVA H S, ALAMINI F, SCHMUTZ J, MORGANTE M, ROKHSAR M D. The high -quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution. Nature Genetica, 2013, 45(5): 487-493. [25] PUIG J, MEYNARD D, KHONG G N, PAULUZZI G, GUIDERDONI E, GANTET P. Analysis of the expression of the AGL17-like clade of MADS-box transcription factors in rice. Gene Expression Patterns, 2013, 13(5): 160-170. [26] WEI B, ZHANG R, GUO J, LIU D, LI A, FAN R, MAO L, ZHANG X. Genome-wide analysis of the MADS-box gene family in Brachypodium distachyon. PLoS ONE, 2014, 9(1): e84781. [27] DUAN W, SONG X, LIU T, HUANG Z, REN J, HOU X, LI Y. Genome-wide analysis of the MADS-box gene family in Brassica rapa (Chinese cabbage). Molecular Genetics and Genomics, 2015, 290(1): 239-255. [28] JACK T, BROCKMAN L L, MEYEROWITZ E M. The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens. Cell, 1992, 68(4): 683-697. [29] CHANG Y, KAO N, LI J, HSU W, LIANG Y, WU J, YANG C. Characterization of the possible roles for B class MADS box genes in regulation of perianth formation in orchid. Plant Physiology, 2010, 152(2): 837-853. [30] XIAO H, WANG Y, LIU D, WANG W, LI X, ZHAO X, XU J, ZHAI W, ZHU L. Functional analysis of the rice AP3 homologue OsMADS16 by RNA interference. Plant Molecular Biology, 2003, 52(5): 957-966. [31] ZAHN L M, LEEBENS-MACK J, MA H, THEISSEN, G. To B or not to B a flower: the role of DEFICIENS and GLOBOSA orthologs in the evolution of the angiosperms. Journal of Heredity, 2005, 96(3): 225-240. [32] AMBROSE B A, LERNER D R, CICERI P, PADILLA C M, YANOFSKY M F, SCHMIDT R J. Molecular and genetic analyses of the silky1 gene reveal conservation in floral organ specification between eudicots and monocots. Molecular Cell, 2000, 5(3): 569-579. [33] ROQUE E, SERWATOWSKA J, CRUZ ROCHINA M, WEN J, MYSORE K S, YENUSH L, BELTRÁN J P, CAÑAS L A. Functional specialization of duplicated AP3-like genes in Medicago truncatula. The Plant Journal, 2013, 73(4): 663-675. [34] HU L, LIU S. Genome-wide analysis of the MADS-box gene family in cucumber. Genome, 2012, 55(3): 245-256. [35] DÍAZ-RIQUELME J, LIJAVETZKY D, MARTÍNEZ-ZAPATER J M, CARMONA M J. Genome-wide analysis of MIKCC-type MADS box genes in grapevine. Plant Physiology, 2009, 149(1): 354-369. [36] ADAMCZYK B J, FERNANDEZ D E. MIKC* MADS domain heterodimers are required for pollen maturation and tube growth in Arabidopsis. Plant Physiology, 2009, 149(4): 1713-1723. [37] LIU Y, CUI S, WU F, YAN S, LIN X, DU X, CHONG K, SCHILING S, THEIßEN G, MENG Z. Functional conservation of MIKC*-type MADS box genes in Arabidopsis and rice pollen maturation. The Plant Cell, 2013, 25(4): 1288-1303. [38] PORTEREIKO M F, LLOYD A, STEFFEN J G, PUNWANI J A, OTSUGA D, DREWS G N. AGL80 is required for central cell and endosperm development in Arabidopsis. The Plant Cell, 2006, 18(8): 1862-1872. [39] KANG I H, STEFFEN J G, PORTEREIKO M F, LLOYD A, DREWS G N. The AGL62 MADS domain protein regulates cellularization during endosperm development in Arabidopsis. The Plant Cell, 2008, 20(3): 635-647. [40] XU Z, ZHANG Q, SUN L, DU D, CHENG T, PAN H, YANG W, WANG J. Genome-wide identification, characterization and expression analysis of the MADS-box gene family in Prunus mume. Molecular Genetics and Genomics, 2014, 289(5): 903-920. [41] SHU Y, YU D, WANG D, GUO D, GUO C. Genome-wide survey and expression analysis of the MADS-box gene family in soybean. Molecular Biology Reports, 2013, 40(6): 3901-3911.