枣E类MADS基因在花和果中的表达及其蛋白互作研究

doi:10.16420/j.issn.0513-353x.2021-0179

摘要/Abstract

摘要：

以‘金丝小枣’花和果实为试材进行E类MADS-box基因的生物信息、表达、亚细胞定位及蛋白互作研究。3个E-type MADS-box基因分别位于SEP1/2、SEP3和SEP4进化支。与其他物种的同源序列相比,E类蛋白在进化中非常保守。3个E类基因在枣的花和果实中均有表达,ZjSEP1/2在花不同发育时期表达比较稳定,ZjSEP3和ZjSEP4在花发育前期高表达,在感染枣疯病的花变叶中3个基因的表达均显著受抑制。3个蛋白均定位于细胞核。酵母单杂交结果表明ZjSEP1/2和ZjSEP3具有转录激活活性,可以作为转录因子调控下游基因,而ZjSEP4不具有此活性。酵母双杂交结果表明3个E类MADS蛋白都可以和A类、C/D类蛋白发生互作,ZjSEP4与ZjSEP1/2均可与自身互作,但ZjSEP3自身不发生互作。综上,3个E类MADS基因在枣花发育中具有重要作用,但功能和调控模式不同,ZjSEP3不能形成同源二聚体,ZjSEP1/2、ZjSEP4均可形成同源二聚体,3个E类蛋白均可与A、C/D类蛋白形成异源二聚体。

关键词: 枣, MADS-box, E-type, 花果发育, 表达分析, 转录激活活性, 蛋白互作

Abstract:

In order to study the function of E-type MADS-box genes in flower and fruit of Chinese jujube（Ziziphus jujuba Mill.）,their bioinformatics analysis,expression pattern,subcellular location and protein interaction in‘Jinsi Xiaozao’were studied systematically. A total of three E-type MADS-box genes were identified in the jujube genome,which are located in the three clades of SEP1/2,SEP3 and SEP4. Compared with homologous sequences of other species,they are very conservative in evolution. Three E-type genes expressed in jujube flowers and fruits. The expression of ZjSEP1/2 was relatively stable at different stages of flower development,ZjSEP3 and ZjSEP4 were highly expressed in the early stage of flower development. After phytoplasma infection,their expressions in phyllody,a typical diseased symptom, were inhibited dramatically. All three proteins were located in the nucleus. According to the results of yeast one-hybrid analysis,ZjSEP1/2 and ZjSEP3 proteins had transcriptional activation activity and could be used as transcription factors to regulate downstream genes,while ZjSEP4 didn’t have this activity. According to the yeast two-hybrid results,all three proteins could interact with A-type and C/D-type proteins. Both ZjSEP4 and ZjSEP1/2 could interact with itself,but ZjSEP3 couldn’t interact with itself. In summary,the three E-type genes play an important role in the development of jujube flowers,but they should have various regulatory modes. ZjSEP3 couldn’t form homodimers,while ZjSEP4 and ZjSEP1/2 could form homodimers. But all of three proteins could form heterodimers with A-type and C/D-type proteins.

Key words: Ziziphus jujuba, MADS-box gene, E-type, flower and fruit development, expression analysis, transcriptional activation, protein interaction

中图分类号:

S665.1

高玮林, 张力曼, 薛超玲, 张垚, 刘孟军, 赵锦. 枣E类MADS基因在花和果中的表达及其蛋白互作研究[J]. 园艺学报, 2022, 49(4): 739-748.

GAO Weilin, ZHANG Liman, XUE Chaoling, ZHANG Yao, LIU Mengjun, ZHAO Jin. Expression of E-type MADS-box Genes in Flower and Fruits and Protein Interaction Analysis in Chinese Jujube[J]. Acta Horticulturae Sinica, 2022, 49(4): 739-748.

图/表 7

表1 本研究中所用引物名称及序列

Table 1 Primer names and sequences used in this study

用途 Usage	基因名称 Gene name	正向序列（5′-3′） Forward primer sequence	反向序列（5′-3′） Reverse primer sequence
q-RT-PCR Quantitative real-time PCR	ZjSEP1/2	GGGGAGAGGTAAGGTGGAG	GGTTGGAGAAGATGATAAGAGC
	ZjSEP3	GGGGAAGACCTTGGACCTTT	AGCAAGTGTTCCTTGCGTTG
	ZjSEP4	GGGGTTCTTCCAGGCCTTAG	ACTTGCTGGGCATGACTTGT
内参 Primers for internal control	ZjACT	AGCCTTCCTGCCAACGAGT	TTGCTTCTCACCCTTGATGC
酵母双杂交 Yeast two- Hybrid（Y2H）	ZjBDZjSEP1/2	ATGGCCATGGAGGCCGATGGGGAGAGGTAAGGTGGA	GGATCCCCGGGAATTTCAAAGCATCCACCCAGGG
	ZjBDZjSEP3	GGATCCCCGGGAATTTCAAAGCATCCACCCAGGG	GGATCCCCGGGAATTTCATGGTAACCATCCTGGCA
	ZjBDZjSEP4	ATGGCCATGGAGGCCGATGGGGAGGGGTAGAGTTGA	GGATCCCCGGGAATTTCAAAGCATCCATCCAGGAA
	ZjADZjSEP1/2	CCATGGAGGCCAGTGATGGGGAGAGGTAAGGTGGA	CACCCGGGTGGAATTTCAAAGCATCCACCCAGGG
	ZjADZjSEP3	CCATGGAGGCCAGTGATGGGAAGAGGTAGAGTGGA	CACCCGGGTGGAATTTCATGGTAACCATCCTGGCA
	ZjADZjSEP4	CCATGGAGGCCAGTGATGGGGAGGGGTAGAGTTGA	CACCCGGGTGGAATTTCAAAGCATCCATCCAGGAA
	ZjADZjMADS46	CCATGGAGGCCAGTGATGGAGTTCCCAAATCAAGCA	CACCCGGGTGGAATTTCAAACAAGTTGGAGAGCTG
	ZjADMADS24	CCATGGAGGCCAGTGATGGGGAGGGGAAGGGTTC	CACCCGGGTGGAATTGCATCCAAGCGGGCAGGAG
亚细胞定位 Subcellular localization	ZjSEP1/2	AGCTCGGGTACCCGGGATGGGGAGAGGTAAGGTGGA	TCTAGAGGATCAAGCATCCACCCAGGG
	ZjSEP3	AGCTCGGGTACCCGGGATGCTCAAAACACTTGAGA	TCTAGAGGATCTGGTAACCATCCTGGCAT
	ZjSEP4	AGCTCGGGTACCCGGGATGGGGAGGGGTAGAGTTGA	TCTAGAGGATCAAGCATCCATCCAGGAA

图1 枣和其他物种的E类蛋白系统进化树 At:拟南芥;Md:苹果;Pp:桃;Pa:樱桃;Ac:猕猴桃;Vv:葡萄;Pyp:沙梨;Os:水稻;Cj:日本柳杉;DAL1:欧洲云杉;CRM6:水蕨;FBP:矮牵牛。

Fig. 1 Phylogenetic tree of E-type proteins from Chinese jujube and other species At:Arabidopsis thaliana;Md:Malus × domestica;Pp:Prunus persica;Pa:Prunus avium;Ac:Actinidia chinensis;Vv:Vitis vinifera;Pyp:Pyrus pyrifolia;Os:Oryza sativa;Cj:Cryptomeria japonica;DAL1:Picea abies;CRM6:Ceratopteris pteridoides;FBP:Petunia hybrida.

图2 枣E类基因在不同发育时期花（A）、果实（B）和四轮花器官（C）中的表达模式根据邓肯氏多组样本差异显著性分析,不同小写字母表示每个基因的表达在花、果实不同发育阶段和不同花器官中差异显著（P < 0.05）。

Fig. 2 Expression pattern of jujube E-type genes in flowers（A）,fruit（B）and four-whorl floral organs（C） According to the Duncan’s multiple range test,the different letters above the column indicate the significant differences in flower,fruit at different development stages and four-wheel flower organs for each gene（P < 0.05）.

图3 枣E类基因在枣疯病花变叶中的表达

Fig. 3 Expression of E-type gene in phyllody of jujube witches broom t-test,** α = 0.01.

图4 枣E类MADS蛋白的亚细胞定位分析

Fig. 4 Subcellular location analysis of E-type proteins in Chinese jujube

图5 枣E类MADS蛋白的转录激活活性

Fig. 5 The transcription activation activity of E-type proteins in Chinese jujube

图6 枣E类MADS蛋白的酵母双杂交筛选

Fig. 6 Yeast two-hybrid results of E-type proteins in Chinese jujube -LW:SD/-Trp/-Leu;-LWH:SD/-Trp/-Leu/-His;-LWAH:SD/-Trp/-Leu/-His/-Ade.

参考文献 34

[1]	Becker A, Theissen G. 2003. The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Molecular Phylogenetics and Evolution, 29 (3):464-489. doi: 10.1016/S1055-7903(03)00207-0 URL
[2]	Coen E S, Meyerowitz E M. 1991. The war of the whorls:genetic interactions controlling flower development. Nature, 353 (6339):31-37. doi: 10.1038/353031a0 URL
[3]	Ditta G, Pinyopich A, Robles P, Pelaz S, Yanofsky M F. 2004. The SEP 4 gene of Arabidopsis thaliana functions in floral organ and meristem identity. Current Biology, 14 (21):1935-1940. pmid: 15530395
[4]	Günter Theißen. 2001. Development of floral organ identity:stories from the MADS house. Current Opinion in Plant Biology, 4 (1):75-85. doi: 10.1016/S1369-5266(00)00139-4 URL
[5]	Honma T, Goto K. 2001. Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature, 409 (6819):525-529. doi: 10.1038/35054083 URL
[6]	Huang Fang. 2007. Cloning and functional analysis of flower development related genes in soybean(Glycine max L.)[Ph. D. Dissertation]. Nanjing: Nanjing Agricultural University. (in Chinese)
	黄方. 2007. 大豆花发育相关基因的克隆与功能研究[博士论文]. 南京: 南京农业大学.
[7]	Huang J, Zhang C, Zhao X, Fei Z, Wan K, Zhang Z, Pang X, Yin X, Bai Y, Sun X, Gao L, Li R, Zhang J, Li X. 2016. The jujube genome provides insights into genome evolution and the domestication of sweetness/acidity taste in fruit trees. PLoS Genetics, 12 (12):e1006433. doi: 10.1371/journal.pgen.1006433 URL
[8]	Immink R G, Tonaco I A, de Folter S, Shchennikova A, van Dijk A D, Busscher-Lange J, Borst J W, Angenent G C. 2009. SEPALLATA3:the‘glue’for MADS box transcription factor complex formation. Genome Biology, 10 (2):R24. doi: 10.1186/gb-2009-10-2-r24 URL
[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. 2013. Apple SEPALLATA1/2-like genes control fruit flesh development and ripening. Plant Journal, 73 (6):1044-1056. doi: 10.1111/tpj.12094 URL
[10]	Kobayashi K, Yasuno N, Sato Y, Yoda M, Yamazaki R, Kimizu M, Yoshida H, Nagamura Y, Kyozuka J. 2012. Inflorescence meristem identity in rice is specified by overlapping functions of three AP 1 /FUL-like MADS box genes and PAP2,a SEPALLATA MADS box gene. Plant Cell, 24 (5):1848-1859. doi: 10.1105/tpc.112.097105 URL
[11]	Krizek B A, Meyerowitz E M. 1996. The Arabidopsis homeotic genes APETALA3 and PISTILLATA are sufficient to provide the B class organ identity function. Development, 122 (1):11-22. pmid: 8565821
[12]	Li Chengru, Dong Na, Li Xiaoping, Wu Shasha, Liu Zhongjian, Zhai Junwen. 2020. A review of MADS-box genes,the molecular regulatory genes for floral organ development in Orchidaceae. Acta Horticulturae Sinica, 47 (10):2047-2062. (in Chinese)
	李成儒, 董钠, 李笑平, 吴沙沙, 刘仲健, 翟俊文. 2020. 兰科植物花发育调控MADS-box基因家族研究进展. 园艺学报, 47 (10):2047-2062.
[13]	Liao Y T, Lin S S, Lin S J, Shen B N, Cheng H P, Lin C P. 2019. Structural insights into the interaction between phytoplasmal effector causing phyllody 1(PHYL1)and MADS transcription factor. Plant Journal, 100 (4):706-719. doi: 10.1111/tpj.14463 URL
[14]	Lin X, Wu F, Du X, Shi X, Liu Y, Liu S, Hu Y, Theissen G, Meng Z. 2014. The pleiotropic SEPALLATA-like gene OsMADS34 reveals that the‘empty glumes’of rice(Oryza sativa)spikelets are in fact rudimentary lemmas. New Phytologist, 202 (2):689-702. doi: 10.1111/nph.12657 URL
[15]	Liu M J, Zhao J, Cai Q L, Liu G C, Wang J R, Zhao Z H, Liu P, Dai L, Yan G, Wang W J, Li X S, Chen Y, Sun Y D, Liu Z G, Lin M J, Xiao J, Chen Y Y, Li X F, Wu B, Ma Y, Jian J B, Yang W, Yuan Z, Sun X C, Wei Y L, Yu L L, Zhang C, Liao S G, He R J, Guang X M, Wang Z, Zhang Y Y, Luo L H. 2014. The complex jujube genome provides insights into fruit tree biology. Nature Communications, 28 (5):5315.
[16]	Liu Shinan, Qi Tiantian, Ma Jingjing, Ma Lyuyi, Lin Xinchun. 2016. Cloning and functional analysis of SEP-like gene from phyllostachys violascens. Journal of Nuclear Agricultural Sciences, 30 (8):1453-1459. (in Chinese)
	刘世男, 戚田田, 马晶晶, 马履一, 林新春. 2016. 雷竹SEP-like基因的克隆及功能分析. 核农学报, 30 (8):1453-1459.
[17]	Ma H, Yanofsky M F, Meyerowitz E M. 1991. AGL1AGL6,an Arabidopsis gene family with similarity to floral hometic and transcription factor genes. Genes and Development, 5 (3):484-495. pmid: 1672119
[18]	MacLean A M, Sugio A, Makarova O V, Findlay K C, Grieve V M. 2011. Phytoplasma effector SAP54 induces indeterminate leaf-like flower development in Arabidopsis plants. Plant Physiology 157:831-841.
[19]	Maejima K, Iwai R, Himeno M, Komatsu K, Kitazawa Y, Fujita N, Ishikawa K, Fukuoka M, Minato N, Yamaji Y, Oshima K, Namba S. 2014. Recognition of floral homeotic MADS domain transcription factors by a phytoplasmal effector,phyllogen,induces phyllody. Plant Journal, 78 (4):541-554. doi: 10.1111/tpj.12495 URL
[20]	Maejima K, Kitazawa Y, Tomomitsu T, Yusa A, Neriya Y, Himeno M, Yamaji Y, Oshima K, Namba S. 2015. Degradation of class E MADs-domain transcription factors in Arabidopsis by a phytoplasmal effector,phyllogen. Plant Signaling and Behavior, 10 (8):e1042635. doi: 10.1080/15592324.2015.1042635 URL
[21]	Melzer R, Verelst W, Theissen G. 2009. The class E floral homeotic protein SEPALLATA 3 is sufficient to loop DNA in‘floral quartet’-like complexes in vitro. Nucleic Acids Research, 37 (1):144-157. doi: 10.1093/nar/gkn900 URL
[22]	Meng X W, Li Y, Yuan Y, Zhang Y, Li H T, Zhao J, Liu M J. 2020. The regulatory pathways of distinct flowering characteristics in Chinese jujube. Horticulture Research, 7 (1):13-19. doi: 10.1038/s41438-019-0236-1 URL
[23]	Ng M, Yanofsky M F. 2001. Function and evolution of the plant MADS box gene family. Nature Reviews Genetics, 2:186-195. pmid: 11256070
[24]	Pelaz S, Ditta G S, Baumann E, Wisman E, Yanofsky M F. 2000. B and C floral organ identity functions require SEPALLATA MADS box genes. Nature, 405 (6783):200-203. doi: 10.1038/35012103 URL
[25]	Pelaz S, Tapia-López R, Alvarez-Buylla E R, Yanofsky M F. 2001. Conversion of leaves into petals in Arabidopsis. Current Biology, 11 (3):182-184. pmid: 11231153
[26]	Seymour G B., Ryder C D, Cevik V, Hammond J P, Popovich A., King G J, Vrebalov J, Giovannoni J J, Manning K. 2011. A SEPALLATA gene is involved in the development and ripening of strawberry(Fragaria × ananassa Duch.)fruit,a non-climacteric tissue. Journal of Experimental Botany, 62 (3):1179-1188. doi: 10.1093/jxb/erq360 pmid: 21115665
[27]	Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6:molecular evolutionary genetics analysis Version 6.0. Molecular Biology & Evolution, 30 (12):2725-2729.
[28]	Teo Z W N, Zhou W, Shen L. 2019. Dissecting the function of MADS-box transcription factors in orchid reproductive development. Frontiers in Plant Science, 15 (10):1474.
[29]	Xu K, Huang X, Wu M, Wang Y, Chang Y, Liu K, Zhang J, Zhang Y, Zhang F, Yi L, Li T, Wang R, Tan G, Li C. 2014a. A rapid,highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis. PLoS ONE, 9 (1):e83556. doi: 10.1371/journal.pone.0083556 URL
[30]	Xu Y, Zhang L, Xie H, Zhang Y Q, Oliveira M M, Ma R C. 2008. Expression analysis and genetic mapping of three SEPALLATA -like genes from peach(Prunus persica(L.)Batsch). Tree Genetics & Genomes, 4 (4):693-703.
[31]	Xu Z, Zhang Q, Sun L, Du D, Cheng T, Pan H, Yang W, Wang J. 2014b. Genome-wide identification,characterisation and expression analysis of the MADS-box gene family in Prunus mume. Molecular Genetics and Genomics, 289 (5):903-920. doi: 10.1007/s00438-014-0863-z URL
[32]	Zhang L M, Zhao J, Feng C F, Liu M J, Wang J R, Hu Y F. 2017. Genome-wide identification,characterization of the MADS-box gene family in Chinese jujube and their involvement in flower development. Scientific Reports, 7 (1):1025. doi: 10.1038/s41598-017-01159-8 URL
[33]	Zhou Y, Xu Z, Yong X, Ahmad S, Yang W, Cheng T, Wang J, Zhang Q. 2017. SEP-class genes in Prunus mume and their likely role in floral organ development. BMC Plant Biology, 17 (1):10. doi: 10.1186/s12870-016-0954-6 URL
[34]	Zong Chengwen, Fang Jinggui, Tao Jianmin, Zhang Zhen, Qian Yaming. 2007. Cloning and sequence analysis of MADS-box family protein gene fragments from grape. Journal of Fruit Science, 25 (1):27-32. (in Chinese)
	宗成文, 房经贵, 陶建敏, 章镇, 钱亚明. 2007. 葡萄MADS-box家族基因保守片段的克隆与序列分析. 果树学报, 25 (1):27-32.