巴西橡胶树HbSIP2互作蛋白的筛选和鉴定
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摘要
可溶性无机焦磷酸酶在天然橡胶生物合成中具有重要的调控作用,HbSIP2是胶乳中关键的可溶性无机焦磷酸酶基因。为了深入了解HbSIP2调控橡胶生物合成的机理,本研究对HbSIP2互作蛋白进行了筛选和鉴定。结果表明:诱饵载体p GBKT7-Hb SIP2无自激活活性,且对酵母无毒性作用,可以用于酵母文库筛选。将诱饵载体与橡胶树胶乳cDNA文库进行杂交,初步筛选获得20个与HbSIP2互作的蛋白。进一步通过双分子荧光互补实验证实,Hb SIP2能够与橡胶延伸因子发生蛋白互作。本研究结果为HbSIP2调控橡胶生物合成的机理研究提供了重要的理论依据。
Soluble inorganic pyrophosphatase plays an important role in natural rubber biosynthesis; HbSIP2 is a key soluble inorganic pyrophosphatase gene in latex. In order to understand the mechanism of HbSIP2 regulating rubber biosynthesis, proteins interacting with HbSIP2 were screened and identified. The results showed that bait plasmid pGBKT7-HbSIP2 had no self-activating activity and no cytotoxicity to the yeast cells, so it can be used to screen yeast library. The bait plasmid pGBKT7-Hb SIP2 was hybridized with latex cDNA library, and 20 proteins interacting with HbSIP2 were obtained. It was further confirmed by bimolecular fluorescence complementation analysis that HbSIP2 could interact with rubber elongation factor. This study might provide an important theoretical basis for further revealing the mechanism of Hb SIP2 regulating rubber biosynthesis.
Soluble inorganic pyrophosphatase plays an important role in natural rubber biosynthesis; HbSIP2 is a key soluble inorganic pyrophosphatase gene in latex. In order to understand the mechanism of HbSIP2 regulating rubber biosynthesis, proteins interacting with HbSIP2 were screened and identified. The results showed that bait plasmid pGBKT7-HbSIP2 had no self-activating activity and no cytotoxicity to the yeast cells, so it can be used to screen yeast library. The bait plasmid pGBKT7-Hb SIP2 was hybridized with latex cDNA library, and 20 proteins interacting with HbSIP2 were obtained. It was further confirmed by bimolecular fluorescence complementation analysis that HbSIP2 could interact with rubber elongation factor. This study might provide an important theoretical basis for further revealing the mechanism of Hb SIP2 regulating rubber biosynthesis.
引文
Beyenbach K.W.,and Wieczorek H.,2006,The V-type H+-AT-Pase:molecular structure and function,physiological roles and regulation,J.Exp.Biol.,209(Pt4):577-589
Bradley M.E.,Rest J.S.,Li W.H.,and Schwartz N.B.,2009,Sulfate activation enzymes:phylogeny and association with pyrophosphatase,Journal of Molecular Evolution,68(1):1-13
Fan W.,Wang H.,Wu Y.,Yang N.,Yang J.,and Zhang P.,2017,H+-pyrophosphatase IbVP1 promotes efficient iron use in sweet potato[Ipomoea batatas(L.)Lam.],Plant Biotechnol.J.,15(6):698-712
Ferjani A.,Segami S.,Horiguchi G.,Muto Y.,Maeshima M.,and Tsukaya H.,2011,Keep an eye on PPi:the vacuolar-type H+-pyrophosphatase regulates postgerminative development in Arabidopsis,The Plant Cell,23(8):2895-2908
Gómez-García M.R.,Losada M.,and Serrano A.,2007,Comparative biochemical and functional studies of family I soluble inorganic pyrophosphatases from photosynthetic bacteria,FEBS J.,274(15):3948-3959
Grzechowiak M.,Sikorski M.,and Jaskolski M.,2013,Inorganic pyrophosphatase(PPase)from a higher plant,BioTechnologia,94(1):35-37
Gutiérrez-Luna F.M.,Sancha E.N.D.L.,Valencia-Turcotte L.G.,Vázquez-Santana S.,and Rodríguez-Sotres R.,2016,Evidence for a non-overlapping subcellular localization of the familyⅠisoforms of soluble inorganic pyrophosphatase in Arabidopsis thaliana,Plant Science,253:229-242
Hu Y.M.,Chen Y.,Qin D.H.,Hu F.R.,and Sun C.B.,2016,Selecting interactor protein of flowering regulatory protein DEF3 in Cymbidium goeringii by yeast two-hybrid system,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(7):1666-1671(胡月苗,陈跃,秦德辉,胡凤荣,孙崇波,2016,利用酵母双杂交系统筛选春兰开花调控蛋白DEF3的互作蛋白,分子植物育种,14(7):1666-1671)
Jacob J.L.,Prév觝t J.C.,Clément-Vidal A.,and D'Auzac J.,1989,Inorganic pyrophosphate metabolism in Hevea brasiliensis latex characteristics of cytosolic alkaline inorganic pyrophosphatase,Plant Physiology and Biochemistry,27:355-364
Klaewklad A.,Nakkanong K.,Nathaworn C.D.,and Nualsri C.,2017,Rubber elongation factor(REF)and small rubber particle protein(SRPP)gene expression responses to variation of seasonal change in four selected rubber clones,Pakistan Journal of Biotechnology,14(1):115-120
Li X.,Guo C.,Gu J.,Duan W.,Zhao M.,Ma C.,Du X.,Lu W.,and Xiao K.,2014,Overexpression of VP,a vacuolar H+-pyrophosphatase gene in wheat(Triticum aestivum L.),improves tobacco plant growth under Pi and N deprivation,high salinity,and drought,J.Exp.Bot.,65(2):683-696
May A.,Berger S.,Hertel T.,and K觟ck M.,2011,The Arabidopsis thaliana phosphate starvation responsive gene AtPPsPase1encodes a novel type of inorganic pyrophosphatase,Biochimica et Biophysica Acta,1810(2):178-185
Priya P.,Venkatachalam P.,and Thulaseedharan A.,2007,Differential expression pattern of rubber elongation factor(REF)m RNA transcripts from high and low yielding clones of rubber tree(Hevea brasiliensis Muell.Arg.),Plant Cell Rep.,26(10):1833-1838
Rodina E.,Vorobieva N.,Kurilova S.,Mikulovich J.,Vainonen J.,Aro E.M.,and Nazarova T.,2011,Identification of new protein complexes of Escherichia coli inorganic pyrophos phatase using pull-down assay,Biochimie,93(9):1576-1583
Schilling R.K.,Marschner P.,Shavrukov Y.,Berger B.,Tester M.,Roy S.J.,and Plett D.C.,2014,Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene(AVP1)improves the shoot biomass of transgenic barley and increases grain yield in a saline field,Plant Biotechnology Journal,12(3):378-386
Siswanto,Prév觝t J.C.,Clément A.,and Jacob J.L.,1994,Characterization of tonoplast pyrophosphatase from Hevea brasiliensis latex,Indian Journal of Natural Rubber Research,7(1):l-8
Zeng R.Z.,Duan C.F.,Li X.Y.,Tian W.M.,and Nie Z.Y.,2009,Vacuolar-type inorganic pyrophosphatase located on the rubber particle in the latex is an essential enzyme in regulation of the rubber biosynthesis in Hevea brasiliensis,Plant Science,176(5):602-607
Bradley M.E.,Rest J.S.,Li W.H.,and Schwartz N.B.,2009,Sulfate activation enzymes:phylogeny and association with pyrophosphatase,Journal of Molecular Evolution,68(1):1-13
Fan W.,Wang H.,Wu Y.,Yang N.,Yang J.,and Zhang P.,2017,H+-pyrophosphatase IbVP1 promotes efficient iron use in sweet potato[Ipomoea batatas(L.)Lam.],Plant Biotechnol.J.,15(6):698-712
Ferjani A.,Segami S.,Horiguchi G.,Muto Y.,Maeshima M.,and Tsukaya H.,2011,Keep an eye on PPi:the vacuolar-type H+-pyrophosphatase regulates postgerminative development in Arabidopsis,The Plant Cell,23(8):2895-2908
Gómez-García M.R.,Losada M.,and Serrano A.,2007,Comparative biochemical and functional studies of family I soluble inorganic pyrophosphatases from photosynthetic bacteria,FEBS J.,274(15):3948-3959
Grzechowiak M.,Sikorski M.,and Jaskolski M.,2013,Inorganic pyrophosphatase(PPase)from a higher plant,BioTechnologia,94(1):35-37
Gutiérrez-Luna F.M.,Sancha E.N.D.L.,Valencia-Turcotte L.G.,Vázquez-Santana S.,and Rodríguez-Sotres R.,2016,Evidence for a non-overlapping subcellular localization of the familyⅠisoforms of soluble inorganic pyrophosphatase in Arabidopsis thaliana,Plant Science,253:229-242
Hu Y.M.,Chen Y.,Qin D.H.,Hu F.R.,and Sun C.B.,2016,Selecting interactor protein of flowering regulatory protein DEF3 in Cymbidium goeringii by yeast two-hybrid system,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),14(7):1666-1671(胡月苗,陈跃,秦德辉,胡凤荣,孙崇波,2016,利用酵母双杂交系统筛选春兰开花调控蛋白DEF3的互作蛋白,分子植物育种,14(7):1666-1671)
Jacob J.L.,Prév觝t J.C.,Clément-Vidal A.,and D'Auzac J.,1989,Inorganic pyrophosphate metabolism in Hevea brasiliensis latex characteristics of cytosolic alkaline inorganic pyrophosphatase,Plant Physiology and Biochemistry,27:355-364
Klaewklad A.,Nakkanong K.,Nathaworn C.D.,and Nualsri C.,2017,Rubber elongation factor(REF)and small rubber particle protein(SRPP)gene expression responses to variation of seasonal change in four selected rubber clones,Pakistan Journal of Biotechnology,14(1):115-120
Li X.,Guo C.,Gu J.,Duan W.,Zhao M.,Ma C.,Du X.,Lu W.,and Xiao K.,2014,Overexpression of VP,a vacuolar H+-pyrophosphatase gene in wheat(Triticum aestivum L.),improves tobacco plant growth under Pi and N deprivation,high salinity,and drought,J.Exp.Bot.,65(2):683-696
May A.,Berger S.,Hertel T.,and K觟ck M.,2011,The Arabidopsis thaliana phosphate starvation responsive gene AtPPsPase1encodes a novel type of inorganic pyrophosphatase,Biochimica et Biophysica Acta,1810(2):178-185
Priya P.,Venkatachalam P.,and Thulaseedharan A.,2007,Differential expression pattern of rubber elongation factor(REF)m RNA transcripts from high and low yielding clones of rubber tree(Hevea brasiliensis Muell.Arg.),Plant Cell Rep.,26(10):1833-1838
Rodina E.,Vorobieva N.,Kurilova S.,Mikulovich J.,Vainonen J.,Aro E.M.,and Nazarova T.,2011,Identification of new protein complexes of Escherichia coli inorganic pyrophos phatase using pull-down assay,Biochimie,93(9):1576-1583
Schilling R.K.,Marschner P.,Shavrukov Y.,Berger B.,Tester M.,Roy S.J.,and Plett D.C.,2014,Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene(AVP1)improves the shoot biomass of transgenic barley and increases grain yield in a saline field,Plant Biotechnology Journal,12(3):378-386
Siswanto,Prév觝t J.C.,Clément A.,and Jacob J.L.,1994,Characterization of tonoplast pyrophosphatase from Hevea brasiliensis latex,Indian Journal of Natural Rubber Research,7(1):l-8
Zeng R.Z.,Duan C.F.,Li X.Y.,Tian W.M.,and Nie Z.Y.,2009,Vacuolar-type inorganic pyrophosphatase located on the rubber particle in the latex is an essential enzyme in regulation of the rubber biosynthesis in Hevea brasiliensis,Plant Science,176(5):602-607