森林草莓FvMOB1与FvM4K1互作及响应生长素调控初探

doi:10.16420/j.issn.0513-353x.2020-0859

摘要/Abstract

摘要：

为研究草莓FvMOB1基因参与生长素调控生长发育及细胞增殖过程中的作用,以森林草莓（Fragraria vesca）‘Hawaii 4’为材料,对同源基因FvMOB1A和FvMOB1B的编码区序列进行基因克隆,对基因进行生物信息分析。应用荧光定量PCR分析了FvMOB1在森林草莓不同器官、不同发育时期果实和IAA处理不同天数的幼苗中的相对表达量,对FvMOB1蛋白进行亚细胞定位和酵母双杂互作分析。结果表明,FvMOB1A和FvMOB1B的开放阅读框分别为648和651 bp,编码215个和216个氨基酸。在森林草莓中,FvMOB1A的表达量远远高于FvMOB1B,其中FvMOB1A在根和瘦果中表达量高,茎中最低,FvMOB1B在瘦果和茎中表达量相对较高,叶中最低;两个基因都在花后30 d（成熟后期）果实中表达最高;幼苗经IAA处理后两个基因的表达模式相同,表达量都显著上调并在7 d达到峰值。FvMOB1A和FvMOB1B蛋白定位在细胞核和细胞膜上,酵母双杂交验证二者均可与促分裂原活化蛋白激酶FvM4K1蛋白互作。

关键词: 森林草莓, MOB1, 生长素, 细胞增殖

Abstract:

In order to study how FvMOB1 genes involved in auxin mediated woodland strawberry growth and regulate cell proliferation,this experiment using woodland strawberry（Fragraria vesca）‘Hawaii 4’material,cloned and analyzed coding sequence of homologous gene FvMOB1A and FvMOB1B based on bioinformatics. Fluorescence quantitative PCR was applied to analysis FvMOB1’s relative expression quantity in different organs of woodland strawberry,different development periods of fruit and different days during IAA treatment. Carried out subcellular localization of FvMOB1 and yeast two-hybrid interaction. The results show that FvMOB1A and FvMOB1B encode 215 and 216 amino acids respectively with open reading frame of 648 and 651 bp. In woodland strawberry,the expression level of FvMOB1A was much higher than that of FvMOB1B,the expression level of FvMOB1A is high in achene and root and lowest in stem,while the expression level of FvMOB1B is relatively highest in achene and lowest in leaves. The expression of both FvMOB1A and FvMOB1B are highest in the 30th day after flowering （post ripening stage）. The expression patterns of the two genes are the same after IAA treatment,both are up-regulated and reaching the peak value on the 7th day. The localization of FvMOB1A and FvMOB1B are in the cell nucleus and cell membrane,and yeast two-hybrid verified that both FvMOB1A and FvMOB1B can interact with mitogen-activated protein kinase FvM4K1.

Key words: woodland strawberry, MOB1, auxin, cell proliferation

中图分类号:

S668.4

刘璐, 谷思, 张伟伟, 赵帅琪, 李安然, 邢宇. 森林草莓FvMOB1与FvM4K1互作及响应生长素调控初探[J]. 园艺学报, 2021, 48(11): 2185-2196.

LIU Lu, GU Si, ZHANG Weiwei, ZHAO Shuaiqi, LI Anran, XING Yu. A Primary Study on FvMOB1 Interact with FvM4K1 and Response to Auxin Regulation in Fragraria vesca[J]. Acta Horticulturae Sinica, 2021, 48(11): 2185-2196.

图/表 9

表1 本试验中所用的引物

Table 1 Primers used in this study

引物名称 Primer name	序列（5′-3′） Sequence
FvMOB1A-F	ATGAGTCTCTTCGGTCTGGG
FvMOB1A-R	GGGTCAATAAGGAACGATGATAGA
FvMOB1B-F	ATGAGTCTCTTTGGTCTTGGAAG
FvMOB1B-R	TCACTTTGGCTGTAGAATAGAGTC
FvMOB1A-QF	TTGATGGACTGGATTGAAT
FvMOB1A-QR	TGAAGCAGGTATTTAGATGG
FvMOB1B-QF	GCAGGACCAAAGTATGAG
FvMOB1B-QR	CACGGAACATTCGCTTAA
FvActin-QF	GGGCCAGAAAGATGCTTATGTCGG
FvActin-QR	GGGCAACACGAAGCTCATTGTAGAAG
FvMOB1A-LF	ATCGACTCTAGAAAGCTTATGAGTCTCTTCGGTCTG
FvMOB1A-LR	CTCCTCGCCCTTGCTCACCATGGTACCATAAGGAACGATGATAGATTCT
FvMOB1B-LF	ATCGACTCTAGAAAGCTTATGAGTCTCTTTGGTCTT
FvMOB1B-LR	CTCCTCGCCCTTGCTCACCATGGTACCCTTTGGCTGTAGAATAGAGTC
FvMOB1A-AF	ATATGGCCATGGAGGCCAGTGAATTCATGAGTCTCTTCGGTCTGG
FvMOB1A-AR	CGATTCATCTGCAGCTCGAGTCAATAAGGAACGATGATAGATTCT
FvMOB1B-AF	ATATGGCCATGGAGGCCAGTGAATTCATGAGTCTCTTTGGTCTTGGAAG
FvMOB1B-AR	TATCTACGATTCATCTGCAGCTCGAGTCACTTTGGCTGTAGAATAGAGTCA
FvM4K1-BF	TGCATATGGCCATGGAGGCCAGTGAATTCATGGCTTTCTTATTCTTCCT
FvM4K1-BR	TGCGGCCGCTGCAGGTCGACGGATCCCTACAAACGAAGAATGGTTCTTA

图1 FvMOB1A和FvMOB1B的基因扩增结果

Fig. 1 The PCR result of FvMOB1A and FvMOB1B genes

图2 不同物种MOB1氨基酸序列比对 Sc:芽殖酵母;At:拟南芥;Fv:森林草莓;Ms:紫花苜蓿;Dm:果蝇;Hs:人。框内为Mob1/phocein family结构域。

Fig. 2 Amino acid sequence alignment of MOB1 in different species Sc:Saccharomyces cerevisiae;At:Arabidopsis thaliana;Fv:Fragraria vesca;Ms:Medicago sativa; Dm:Drosophila melanogaster;Hs:Homo sapiens. Frame means Mob1/phocein family domain.

图3 基于氨基酸序列构建的MOB1基因系统发育树

Fig. 3 Phylogenetic tree of MOB1 gene based on amino acid

图4 不同器官中FvMOB1相对表达量不同小写字母表示差异显著（P < 0.05）。

Fig. 4 Relative expression of FvMOB1 in different organs Different lowercase letters indicate significant differences（P < 0.05）.

图5 果实不同时期FvMOB1相对表达量不同小写字母表示差异显著（P < 0.05）。

Fig. 5 Relative expression of FvMOB1 in different development stages of fruit Different lowercase letters indicate significant differences（P < 0.05）.

图6 IAA 处理下森林草莓幼苗FvMOB1相对表达量不同小写字母表示差异显著（P < 0.05）。

Fig. 6 Relative expression of FvMOB1 in seedlings of woodland strawberry after IAA treatment Different lowercase letters indicate significant differences（P < 0.05）.

图7 FvMOB1A和FvMOB1B蛋白的亚细胞定位

Fig. 7 Subcellular localization of protein FvMOB1A and FvMOB1B

图8 酵母双杂验证FvMOB1与FvM4K1互作

Fig. 8 Yeast two-hybrid verification the interaction of FvMOB1 and FvM4K1

参考文献 35

[1]	Bichsel S J, Tamaskovic R, Stegert M R, Hemmings B A. 2004. Mechanism of activation of NDR(nuclear Dbf2-related)protein kinase by the hMOB1 protein. Journal of Biological Chemistry, 279:35228-35235. pmid: 15197186
[2]	Bögre L, Calderini O, Binarova P, Mattauch M, Till S, Kiegerl S, Jonak C, Pollaschek C, Huskisson N S, Hirt H, Heberle-Bors E. 1999. A MAP kinase is activated late in plant mitosis and becomes localized to the plane of cell division. Plant Cell, 11:101-113. pmid: 9878635
[3]	Brumos J, Robles L M, Yun J, Vu T C, Jackson S, Alonso J M, Stepanova A N. 2018. Local auxin biosynthesis is a key regulator of plant development. Developmental Cell, 47:306-318. doi: 10.1016/j.devcel.2018.09.022 URL
[4]	Citterio S, Albertini E, Varotto S, Feltrin E, Soattin M, Marconi G, Sgorbati S, Lucchin M, Barcaccia G. 2005. Alfalfa Mob1-like genes are expressed in reproductive organs during meiosis and gametogenesis. Plant Molecular Biology, 58:789-807. doi: 10.1007/s11103-005-8104-9 pmid: 16240174
[5]	Citterio S, Piatti S, Albertini E, Aina R, Varotto S, Barcacci G. 2006. Alfalfa Mob1-like proteins are involved in cell proliferation and are localized in the cell division plane during cytokinesis. Experimental Cell Research, 312:1050-1064. pmid: 16460730
[6]	Cui X N, Guo Z A, Song L Z, Wang Y L, Cheng Y F. 2016. NCP1/AtMOB1A plays key roles in auxin-mediated Arabidopsis development. PLoS Genetics, 12:e1005923.
[7]	Galla G, Zenoni S, Marconi G, Marino G, Botton A, Pinosa A, Citterio S, Ruperti B, Palme K, Albertini E, Pezzotti M, Mau M, Sharbel T F, Storme N D, Geelen D, Barcacci G. 2011. Sporophytic and gametophytic functions of the cell cycle-associated Mob1 gene in Arabidopsis thaliana L. Gene, 484:1-12. doi: 10.1016/j.gene.2011.05.009 URL
[8]	Gu Si, Cao Xiao-yan, Xing Yu. 2020. Cloning and expression analysis of FvM4K1 in Fragaria vesca. Journal of Beijing University of Agriculture, 35 (3):8-12. (in Chinese)
	谷思, 曹小艳, 邢宇. 2020. 森林草莓FvM4K1基因的克隆及表达分析. 北京农学院学报, 35 (3):8-12.
[9]	Guo Z A, Yue X Z, Cui X N, Song L Z, Cheng Y F. 2020. AtMOB 1 genes regulate jasmonate accumulation and plant development. Plant Physiology, 182:1481-1493. doi: 10.1104/pp.19.01434 URL
[10]	Harvey K F, Pfleger C M, Hariharan I K. 2003. The Drosophila Mst ortholog,hippo,restricts growth and cell proliferation and promotes apoptosis. Cell, 114:457-467. doi: 10.1016/S0092-8674(03)00557-9 URL
[11]	Hergovich A, Bichsel S J, Hemmings B A. 2005. Human NDR kinases are rapidly activated by MOB proteins through recruitment to the plasma membrane and phosphorylation. Molecular and Cellular Biology, 25:8259-8272. doi: 10.1128/MCB.25.18.8259-8272.2005 URL
[12]	Hou Y X, Wang X T, Zhu Z J, Sun M X, Li M L, Hou L P. 2020. Expression analysis of genes related to auxin metabolism at different growth stages of pak choi. Horticultural Plant Journal, 6 (1):25-33. doi: 10.1016/j.hpj.2019.12.003 URL
[13]	Johnson G L, Lapadat R. 2002. Mitogen-activated protein kinase pathways mediated by ERK,JNK,and p38 protein kinases. Science, 298:1911-1912. pmid: 12471242
[14]	Lai Z, Wei X, Shimizu T, Ramos E, Rohrbaugh R, Nikolaidis N, Lun H, Ying L. 2005. Control of cell proliferation and apoptosis by Mob1 as tumor suppressor. Cell, 120:675-685. doi: 10.1016/j.cell.2004.12.036 URL
[15]	Long Qin, Xie Yu, Lei Tiangang, He Yongrui, Xu Lanzhen, Zou Xiuping, Chen Shanchun. 2020. Bioinformatics analysis of MAPK genes in citrus and their expression in response to canker disease. Acta Horticulturae Sinica, 47 (11):2095-2106. (in Chinese)
	龙琴, 谢宇, 雷天刚, 何永睿, 许兰珍, 邹修平, 陈善春. 2020. 柑橘MAPK基因的生物信息及其响应溃疡病菌侵染的表达分析. 园艺学报, 47 (11):2095-2106.
[16]	Luca F C, Mody M, Kurischko C, Roof D M, Giddings T H, Winey M. 2001. Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit. Molecular and Cellular Biology, 20:6972-6983.
[17]	Luca F C, Winey M. 1998. MOB1,an essential yeast gene required for completion of mitosis and maintenance of ploidy. Molecular and Cellular Biology, 9:29-46.
[18]	Mah A.S. Jang J. Deshaies R J. 2001. Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex. PNAS, 98 (13):7325-7330. pmid: 11404483
[19]	Overvoorde P, Fukaki H, Beeckman T. 2010. Auxin control of root development. Cold Spring Harbor Perspectives Biology, 2:a001537.
[20]	Pan D. 2010. The hippo signaling pathway in development and cancer. Dev Cell, 19:491-505. doi: 10.1016/j.devcel.2010.09.011 URL
[21]	Pinosa F, Begheldo M, Pasternak T, Zermiani M, Papono I A, Dovzhenko A, Barcaccia G, Ruperti B, Palme K. 2013. The Arabidopsis thaliana Mob1A gene is required for organ growth and correct tissue patterning of the root tip. Annals of Botany, 112:1803-1814. doi: 10.1093/aob/mct235 URL
[22]	Ramazan G, Alexander H. 2019. MOB(Mps one Binder)proteins in the hippo pathway and cancer. Cells, 8:569-596. doi: 10.3390/cells8060569 URL
[23]	Ramos A, Camargo F D. 2012. The Hippo signaling pathway and stem cell biology. Trends in Cell Biology, 22:339-346. doi: 10.1016/j.tcb.2012.04.006 URL
[24]	Rawat S J, Chernoff J. 2015. Regulation of Mammalian Ste20(Mst)kinases. Trends in Biochemical Sciences, 40 (3):149-156. doi: 10.1016/j.tibs.2015.01.001 URL
[25]	Shimizu T, Ho L, Lai Z. 2008. The mob as tumor suppressor gene is essential for early development and regulates tissue growth in Drosophila. Genetics, 178:957-965. doi: 10.1534/genetics.107.081570 URL
[26]	Vander B E A, Jones J D G. 1998. The NB-ARC domain:a novel signaling motif shared by plant resistance gene products and regulators of cell death in animals. Current Biology, 8:226-227.
[27]	Vanneste S, Friml J. 2009. Auxin:a trigger for change in plant development. Cell, 136:1005-1016. doi: 10.1016/j.cell.2009.03.001 pmid: 19303845
[28]	Wu S A, Huang J B, Dong J X, Pan D J. 2003. Hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts. Cell, 114:445-456. doi: 10.1016/S0092-8674(03)00549-X URL
[29]	Xiong J, Cui X, Yuan X, Yu X, Sun J, Gong Q. 2015. The Hippo/STE20 homolog SIK1 interacts with MOB1 to regulate cell proliferation and cell expansion in Arabidopsis. Journal of Experimental Botany, 67:1461-1475. doi: 10.1093/jxb/erv538 URL
[30]	Yan Shuangshuang, Qiu Zhengkun, Yu Bingwei, Ming Fangyan, Chen Changming, Lei Jianjun, Cao Bihao. 2020. Advances in phytohormone auxin response to high temperature. Acta Horticulturae Sinica, 47 (11):2238-2246. (in Chinese)
	颜爽爽, 邱正坤, 余炳伟, 明方艳, 陈长明, 雷建军, 曹必好. 2020. 植物生长素响应高温胁迫研究进展. 园艺学报, 47 (11):2238-2246. (in Chinese)
[31]	Zeng W, Dai X, Sun J, Hou Y, Ma X, Cao X, Zhao Y, Cheng Y. 2018. Modulation of auxin signaling and development by polyadenylation machinery. Plant Physiology, 179:686-699. doi: 10.1104/pp.18.00782 URL
[32]	Zhang M, Chiang Y, Toruno T, Lee D, Ma M, Liang X, Lal N K, Lemos M, Lu Y, Ma S. 2018. The MAP4 kinase SIK1 ensures robust extracellular ROS burst and antibacterial immunity in plants. Cell Host Microbe, 24:379-391. doi: 10.1016/j.chom.2018.08.007 URL
[33]	Zhang Ping-ping, Tong Xin, Zhang Tian-le, Li Zi-chen, Gong Qing-qiu. 2017. The emerging Hippo signaling pathway in plants. Hereditas, 39 (7):568-575. (in Chinese)
	张平平, 佟鑫, 张天乐, 黎子琛, 龚清秋. 2017. 植物Hippo信号通路研究进展. 遗传, 39 (7):568-575.
[34]	Zhao B, Tumaneng K, Guan K. L. 2011. The Hippo pathway in organ size control,tissue regeneration and stem cell self-renewal. Nature Cell Biology, 13:877-883.
[35]	Zhou H Y, Ren S Y, Han Y F, Zhang Q, Qin L, Xing Y. 2017. Identification and analysis of mitogen-activated protein kinase (MAPK)cascades in Fragaria vesca. International Journal of Molecular Sciences, 18 (8):1766. doi: 10.3390/ijms18081766 URL