基于家族基因分析的甘蓝MLPK互作PUB蛋白的筛选
|
摘要
MLPK(M–位点受体激酶)是芸薹属自交不亲和正向调控关键元件,其参与自交不亲和信号传导的分子机制尚不明确,同时自交不亲和下游信号元件也有待于进一步分离。为了探索分离MLPK互作蛋白的思路和方法,构建了不含核定位信号的MLPK短截蛋白(MLPK-T),并利用酵母双杂交检测到MLPK与臂重复蛋白1(ARC1)作用,通过全基因组鉴定分别获得了96个甘蓝、101个白菜、70个琴叶拟南芥和62个拟南芥PUB蛋白,其中含有臂重复序列的PUB蛋白共为127个。通过系统进化分析,筛选到8个含臂重复序列的甘蓝BoPUB蛋白,其8个基因全部在柱头内表达,且成功利用酵母双杂交检测到MLPK与3个含臂重复序列的BoPUB蛋白Bol008579、Bol016165和Bol023511相互作用。
MLPK(M-locus protein kinase)is a positive key mediator of Brassica self-incompatibility signaling,however,the molecular mechanism of MLPK in mediating Brassica self-incompatibility response is still unknown. Meanwhile,the downstream factors of Brassica self-incompatibility signaling still need to be isolated. Aim to explore the idea and methods to isolate MLPK-interacting proteins,here,the MLPK truncated protein MLPK-T lack of the plasma membrane localization motif was constructed,the interaction of MLPK with ARC1 was proved via conventional yeast two-hybrid system. Then,we obtained 96,101,70 and 62 PUB proteins from B. olercea,B. rapa,Arabidopsis lyrata and A. thaliana through genome-wide identification,amount to 127 PUB proteins containing ARM repeat domains. Based on phylogenetic analysis,we selected 8 PUB proteins,and RT-PCR showed that all these 8 genes expressed in B. oleracea stigmas. Finally,the interaction of MLPK with Bol008579,Bol016165 and Bol023511 were proved through yeast two-hybrid system.
MLPK(M-locus protein kinase)is a positive key mediator of Brassica self-incompatibility signaling,however,the molecular mechanism of MLPK in mediating Brassica self-incompatibility response is still unknown. Meanwhile,the downstream factors of Brassica self-incompatibility signaling still need to be isolated. Aim to explore the idea and methods to isolate MLPK-interacting proteins,here,the MLPK truncated protein MLPK-T lack of the plasma membrane localization motif was constructed,the interaction of MLPK with ARC1 was proved via conventional yeast two-hybrid system. Then,we obtained 96,101,70 and 62 PUB proteins from B. olercea,B. rapa,Arabidopsis lyrata and A. thaliana through genome-wide identification,amount to 127 PUB proteins containing ARM repeat domains. Based on phylogenetic analysis,we selected 8 PUB proteins,and RT-PCR showed that all these 8 genes expressed in B. oleracea stigmas. Finally,the interaction of MLPK with Bol008579,Bol016165 and Bol023511 were proved through yeast two-hybrid system.
引文
Bower M S,Matias D D,Fernandes-Carvalho E,Mazzurco M,Gu T,Rothstein S J,Goring D R.1996.Two members of the thioredoxin-h family interact with the kinase domain of a Brassica S locus receptor kinase.Plant Cell,8(9):1641-1650.
Doucet J,Lee H K,Goring D R.2016.Pollen acceptance or rejection:a tale of two pathways.Trends in Plant Science,21(12):1058-1067.
Gao Q G,Shi S M,Liu Y D,Pu Q M,Liu X H,Zhang Y,Zhu L Q.2016.Identification of a novel MLPK homologous gene MLPKn1 and its expression analysis in Brassica oleracea.Plant Reproduction,29(3):239-250.
Goring D R,Glavin T L,Schafer U,Rothstein S J.1993.An S receptor kinase gene in self-compatible Brassica napus has a 1-bp deletion.Plant Cell,5(5):531-539.
Goring D R,Indriolo E,Samuel M A.2014.The ARC1 E3 ligase promotes a strong and stable self-Incompatibility response in Arabidopsis species:response to the Nasrallah and Nasrallah commentary.Plant Cell,26(10):3842-3846.
Gu T,Mazzurco M,Sulaman W,Matias D D,Goring D R.1998.Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase.Proceedings of the National Academy of Sciences,95(1):382-387.
Haasen K E,Goring D R.2010.The recognition and rejection of self-incompatible pollen in the Brassicaceae.Botanical Studies,51(1):1-6.
Indriolo E,Goring D R.2016.Yeast two-hybrid interactions between Arabidopsis lyrata S Receptor Kinase and the ARC1 E3 ligase.Plant Signal and Behavior,11(6):e1188233.
Indriolo E,Tharmapalan P,Wright SI,Goring D R.2012.The ARC1 E3 ligase gene is frequently deleted in self-compatible Brassicaceae species and has a conserved role in Arabidopsis lyrata self-pollen rejection.Plant Cell,24(11):4607-4620.
Kachroo A,Nasrallah M E,Nasrallah J B.2002.Self-incompatibility in the Brassicaceae:receptor-ligand signaling and cell-to-cell communication.Plant Cell,14:S227-S238.
Kakita M,Murase K,Iwano M,Matsumoto T,Wantanabe M,Shiba H,Isogai A,Takayama S.2007a.Two distinct forms of M-locus protein kinase localize to the plasma membrane and interact directly with S-locus receptor kinase to transduce self-incompatibility signaling in Brassica rapa.Plant Cell,19(12):3961-3973.
Kakita M,Shimosato H,Murase K,Isogai A,Takayama S.2007b.Direct interaction between S-locus receptor kinase and M-locus protein kinase involved in Brassica self-incompatibility signaling.Plant Biotechnology,24(2):185-190.
Kinoshita A,ten Hove C A,Tabata R,Yamada M,Shimizu N,Ishida T,Yamaguchi K,Shigenobu S,Takebayashi Y,Iuchi S,Kobayashi M,Kurata T,Wada T,Seo M,Hasebe M,Blilou I,Fukuda H,Scheres B,Heidstra R,Kamiya Y,Sawa S.2015.A plant U-box protein,PUB4,regulates asymmetric cell division and cell proliferation in the root meristem.Development,142(3):444-453.
Kitashiba H,Liu P,Nishio T,Nasrallah J B,Nasrallah M E.2011.Functional test of Brassica self-incompatibility modifiers in Arabidopsis thaliana.Proceedings of the National Academy of Sciences,108(44):18173-18178.
Murase K,Shiba H,Iwano M,Che F S,Watanabe M,Isogai A,Takayama S.2004.A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling.Science,303(5663):1516-1519.
Samuel M A,Mudgil Y,Salt J N,Delmas F,Ramachandran S,Chilelli A,Goring D R.2008.Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis.Plant Physiology,147(4):2084-2095.
Stone S L,Arnoldo M,Goring D R.1999.A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants.Science,286(5445):1729-1731.
Tantikanjana T,Nasrallah M E,Nasrallah J B.2010.Complex networks of self-incompatibility signaling in the Brassicaceae.Current Opinion Plant Biology,13(5):520-526.
Vanoosthuyse V,Tichtinsky G,Dumas C,Gaude T,Cock J M.2003.Interaction of calmodulin,a sorting nexin and kinase-associated protein phosphatase with the Brassica oleracea S locus receptor kinase.Plant Physiology,133(2):919-929.
Wang Y P,Wu Y Y,Yu B Y,Yin Z,Xia Y J.2017.EXTRA-LARGE G PROTEINs interact with E3 ligases PUB4 and PUB2 and function in cytokinin and developmental processes.Plant Physiology,173(2):1235-1246.
Watanabe M,Suwabe K,Suzuki G.2012.Molecular genetics,physiology and biology of self-incompatibility in Brassicaceae.Proceedings of the National Academy of Sciences,88(10):519-535.
Zhang J F,Rea A C,Fu T D,Ma C Z,Nasrallah J B.2014.Exploring the role of a stigma-expressed plant U-box gene in the pollination responses of transgenic self-incompatible Arabidopsis thaliana.Plant Reproduction,27(2):59-68.
Doucet J,Lee H K,Goring D R.2016.Pollen acceptance or rejection:a tale of two pathways.Trends in Plant Science,21(12):1058-1067.
Gao Q G,Shi S M,Liu Y D,Pu Q M,Liu X H,Zhang Y,Zhu L Q.2016.Identification of a novel MLPK homologous gene MLPKn1 and its expression analysis in Brassica oleracea.Plant Reproduction,29(3):239-250.
Goring D R,Glavin T L,Schafer U,Rothstein S J.1993.An S receptor kinase gene in self-compatible Brassica napus has a 1-bp deletion.Plant Cell,5(5):531-539.
Goring D R,Indriolo E,Samuel M A.2014.The ARC1 E3 ligase promotes a strong and stable self-Incompatibility response in Arabidopsis species:response to the Nasrallah and Nasrallah commentary.Plant Cell,26(10):3842-3846.
Gu T,Mazzurco M,Sulaman W,Matias D D,Goring D R.1998.Binding of an arm repeat protein to the kinase domain of the S-locus receptor kinase.Proceedings of the National Academy of Sciences,95(1):382-387.
Haasen K E,Goring D R.2010.The recognition and rejection of self-incompatible pollen in the Brassicaceae.Botanical Studies,51(1):1-6.
Indriolo E,Goring D R.2016.Yeast two-hybrid interactions between Arabidopsis lyrata S Receptor Kinase and the ARC1 E3 ligase.Plant Signal and Behavior,11(6):e1188233.
Indriolo E,Tharmapalan P,Wright SI,Goring D R.2012.The ARC1 E3 ligase gene is frequently deleted in self-compatible Brassicaceae species and has a conserved role in Arabidopsis lyrata self-pollen rejection.Plant Cell,24(11):4607-4620.
Kachroo A,Nasrallah M E,Nasrallah J B.2002.Self-incompatibility in the Brassicaceae:receptor-ligand signaling and cell-to-cell communication.Plant Cell,14:S227-S238.
Kakita M,Murase K,Iwano M,Matsumoto T,Wantanabe M,Shiba H,Isogai A,Takayama S.2007a.Two distinct forms of M-locus protein kinase localize to the plasma membrane and interact directly with S-locus receptor kinase to transduce self-incompatibility signaling in Brassica rapa.Plant Cell,19(12):3961-3973.
Kakita M,Shimosato H,Murase K,Isogai A,Takayama S.2007b.Direct interaction between S-locus receptor kinase and M-locus protein kinase involved in Brassica self-incompatibility signaling.Plant Biotechnology,24(2):185-190.
Kinoshita A,ten Hove C A,Tabata R,Yamada M,Shimizu N,Ishida T,Yamaguchi K,Shigenobu S,Takebayashi Y,Iuchi S,Kobayashi M,Kurata T,Wada T,Seo M,Hasebe M,Blilou I,Fukuda H,Scheres B,Heidstra R,Kamiya Y,Sawa S.2015.A plant U-box protein,PUB4,regulates asymmetric cell division and cell proliferation in the root meristem.Development,142(3):444-453.
Kitashiba H,Liu P,Nishio T,Nasrallah J B,Nasrallah M E.2011.Functional test of Brassica self-incompatibility modifiers in Arabidopsis thaliana.Proceedings of the National Academy of Sciences,108(44):18173-18178.
Murase K,Shiba H,Iwano M,Che F S,Watanabe M,Isogai A,Takayama S.2004.A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling.Science,303(5663):1516-1519.
Samuel M A,Mudgil Y,Salt J N,Delmas F,Ramachandran S,Chilelli A,Goring D R.2008.Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis.Plant Physiology,147(4):2084-2095.
Stone S L,Arnoldo M,Goring D R.1999.A breakdown of Brassica self-incompatibility in ARC1 antisense transgenic plants.Science,286(5445):1729-1731.
Tantikanjana T,Nasrallah M E,Nasrallah J B.2010.Complex networks of self-incompatibility signaling in the Brassicaceae.Current Opinion Plant Biology,13(5):520-526.
Vanoosthuyse V,Tichtinsky G,Dumas C,Gaude T,Cock J M.2003.Interaction of calmodulin,a sorting nexin and kinase-associated protein phosphatase with the Brassica oleracea S locus receptor kinase.Plant Physiology,133(2):919-929.
Wang Y P,Wu Y Y,Yu B Y,Yin Z,Xia Y J.2017.EXTRA-LARGE G PROTEINs interact with E3 ligases PUB4 and PUB2 and function in cytokinin and developmental processes.Plant Physiology,173(2):1235-1246.
Watanabe M,Suwabe K,Suzuki G.2012.Molecular genetics,physiology and biology of self-incompatibility in Brassicaceae.Proceedings of the National Academy of Sciences,88(10):519-535.
Zhang J F,Rea A C,Fu T D,Ma C Z,Nasrallah J B.2014.Exploring the role of a stigma-expressed plant U-box gene in the pollination responses of transgenic self-incompatible Arabidopsis thaliana.Plant Reproduction,27(2):59-68.