芒果无机焦磷酸酶基因的克隆及其表达载体构建
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摘要
无机焦磷酸酶(inorganic pyrophosphatase,PPase)催化焦磷酸(PPi)水解为2个无机正磷酸(Pi),是蔗糖合成途径中的调控关键节点之一。本研究根据已经报道的PPase基因的序列设计兼并引物,采用3′RACE和5′RACE方法,从贵妃芒果的果实中克隆得到了一个芒果PPase基因,将其命名为MiPPase,其全长cDNA序列为1014 bp,开放阅读框为837bp,编码278个氨基酸,分子量为30.85ku,等电点为4.68。通过系统发育分析发现该基因编码的蛋白与红花烟草具有较近的亲缘关系。为深入探究MiPPase基因在蔗糖代谢过程中的作用,本研究成功构建出pGreenII62-SK-MiPPase基因过量表达载体,为后续研究MiPPase基因在芒果果实蔗糖合成的作用机理提供理论依据。
The hydrolysis of pyrophosphoric acid(PPi) is catalyzed by inorganic pyrophosphate phosphatase(PPase)into two inorganic orthophosphoric acid(Pi),which is one of the key regulatory nodes in the sucrose synthesis pathway.According to the reported sequence of PPase gene, primers were designed in this study. PPase gene was cloned in the fruit of Guifei mango using the methods of 3′ Race and 5′ Race, and named MiPPase. Its full-length of the cDNA sequence was 1014 bp and the open reading frame was 837 bp. It encoded 278 amino acids with molecular weight of 30.85 ku and its isoelectric point was 4.68. Phylogenetic analysis showed that the protein encoded by the PPase gene was closely related to Nicotiana tabacum. In order to investigate the role of MiPPase gene in sucrose metabolism, the pGreenII 62-SK-MiPPase gene overexpression vector was successfully constructed, which would provide a theoretical basis for the further study of the mechanism of MiPPase gene on the sucrose synthesis in mango fruit.
The hydrolysis of pyrophosphoric acid(PPi) is catalyzed by inorganic pyrophosphate phosphatase(PPase)into two inorganic orthophosphoric acid(Pi),which is one of the key regulatory nodes in the sucrose synthesis pathway.According to the reported sequence of PPase gene, primers were designed in this study. PPase gene was cloned in the fruit of Guifei mango using the methods of 3′ Race and 5′ Race, and named MiPPase. Its full-length of the cDNA sequence was 1014 bp and the open reading frame was 837 bp. It encoded 278 amino acids with molecular weight of 30.85 ku and its isoelectric point was 4.68. Phylogenetic analysis showed that the protein encoded by the PPase gene was closely related to Nicotiana tabacum. In order to investigate the role of MiPPase gene in sucrose metabolism, the pGreenII 62-SK-MiPPase gene overexpression vector was successfully constructed, which would provide a theoretical basis for the further study of the mechanism of MiPPase gene on the sucrose synthesis in mango fruit.
引文
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[15]Gaxiola R A,Li J,Undurraga S,et al.Drought-and salt-tolerant plants result from overexpression of the AVP1H+-pump[J].Proceedings of the National Academy of Sciencens of the United States of America,2001,98(20):11444-11449.
[16]Brini F,Gaxiola R G,Masmoudi K.Cloning and characterization of a wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump[J].Plant Physiology&Biochemistry,2005,43(4):347-354.
[17]Kunite M.Kunitz M.Hydrolysis of adenosine triphosphate by crystalline yeast pyrophosphatase.Effect of zinc and magnesium ions[J].Journal of General Physiology,1962,45(4):31-46.
[18]Lahti R,Pitk?ranta T,Valve E,et al.Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12[J].Journal of Bacteriology,1988,170(12):5901-5907.
[19]Visser K,Heimovaara-Dijkstra S,Kijne J W,et al.Molecular cloning and characterization of an inorganic pyrophosphatase from barley[J].Plant Molecular Biology,1998,37(1):131-140.
[20]刘昂,吴文卫,赵磊,等.氯化镁预处理对美人蕉根部质膜H+-ATPase活性与农田废水硝态氮吸收效率的影响[J].西北植物学报,2014,34(1):128-134.
[21]章英才,陈亚萍,景红霞,等.‘灵武长枣’果实糖积累与蔗糖代谢相关酶的关系[J].果树学报,2014,31(2):250-257.
[22]李明.苹果果实中苹果酸代谢关键酶基因的克隆和表达分析[D].泰安:山东农业大学,2007.
[2]Saha S,Mina B L,Gopinath K A,et al.Relative changes in phosphatase activities as influenced by source and application rate of organic composts in field crops[J].Bioresource Technology,2008,99(6):1750-1757.
[3]George G M,Merwe M J V D,Nunes-Nesi A,et al.Virusinduced gene silencing of plastidial soluble inorganic pyrophosphatase impairs essential leaf anabolic pathways and reduces drought stress tolerance in Nicotiana benthamiana[J].Plant Physiology,2010,154(1):55-56.
[4]Soongyu K,Yiyuong L F,Ruchuan V,et al.Proton pumping inorganic pyrophosphatase of endoplasmic reticulumenriched vesicles from etiolated mung bean seedlings[J].Journal of Plant Physiology,2005,162(2):129-138.
[5]Zeng R,Duan C F,Li X Y,et al.Vacuolar-type inorganic pyrophos-phatase located on the rubber particle in the latex is an essential enzyme in regulation of the rubber biosynthesis in Hevea brasiliensis[J].Plant Science,2009,176(5):602-607.
[6]Zhang J,Li J,Wang X,et al.OVP1,a vacuolar H+-translocating inorganic pyrophosphatase(V-PPase),overexpression improved rice cold tolerance[J].Plant Physiology&Biochemistry,2011,49(1):33-38.
[7]May A,Berger S,Hertel T,et al.The Arabidopsis thaliana phosphate starvation responsive gene AtPPsPase1 encodes a novel type of inorganic pyrophosphatase[J].BBA-General Subjects,2011,1810(2):178-185.
[8]Pace D A,Fang J,Cintron R,et al.Overexpression of a cytosolic pyrophosphatase(Tg PPase)reveals a regulatory role of PP(i)in glycolysis for Toxoplasma gondii[J].Biochemical Journal,2011,440(2):229-240.
[9]Gómezgarcía M R,Losada M,Serrano A.Comparative biochemical and functional studies of family I soluble inorganic pyrophosphatases from photosynthetic bacteria[J].Febs Journal,2007,274(15):3948-3959.
[10]Hernández-Domíguez E E,Valencia-Turcotte L G,RodríguezSotres R.Changes in expression of soluble inorganic pyrophosphatases of Phaseolus vulgaris under phosphate starvation[J].Plant Science,2012,187(4):39-48.
[11]Wang C S,Jiang Q T,Jian M A,et al.Characterization and expression analyses of the H+-pyrophosphatase gene in rye[J].Journal of Genetics,2016,95(3):565-572.
[12]Zhang M,Fang Y,Liang Z,et al.Enhanced expression of vacuolar H+-ATPase subunit E in the roots is associated with the adaptation of Broussonetia papyriferato salt stress[J].Plos One,2012,7(10):e48183.
[13]Nordbo E,Luoto H H,Baykov A A,et al.Two independent evolutionary routes to Na+/H+co-transport function in membrane pyrophosphatases[J].Biochemical Journal,2016,473(19):3099-3111.
[14]Yoon H S,Kim S Y,Kim I S.Stress response of plant H+-PPase-expressing transgenic Escherichia coli and Saccharomyces cerevisiae:a potentially useful mechanism for the development of stress-tolerant organisms[J].Journal of Applied Genetics,2013,54(1):129-133.
[15]Gaxiola R A,Li J,Undurraga S,et al.Drought-and salt-tolerant plants result from overexpression of the AVP1H+-pump[J].Proceedings of the National Academy of Sciencens of the United States of America,2001,98(20):11444-11449.
[16]Brini F,Gaxiola R G,Masmoudi K.Cloning and characterization of a wheat vacuolar cation/proton antiporter and pyrophosphatase proton pump[J].Plant Physiology&Biochemistry,2005,43(4):347-354.
[17]Kunite M.Kunitz M.Hydrolysis of adenosine triphosphate by crystalline yeast pyrophosphatase.Effect of zinc and magnesium ions[J].Journal of General Physiology,1962,45(4):31-46.
[18]Lahti R,Pitk?ranta T,Valve E,et al.Cloning and characterization of the gene encoding inorganic pyrophosphatase of Escherichia coli K-12[J].Journal of Bacteriology,1988,170(12):5901-5907.
[19]Visser K,Heimovaara-Dijkstra S,Kijne J W,et al.Molecular cloning and characterization of an inorganic pyrophosphatase from barley[J].Plant Molecular Biology,1998,37(1):131-140.
[20]刘昂,吴文卫,赵磊,等.氯化镁预处理对美人蕉根部质膜H+-ATPase活性与农田废水硝态氮吸收效率的影响[J].西北植物学报,2014,34(1):128-134.
[21]章英才,陈亚萍,景红霞,等.‘灵武长枣’果实糖积累与蔗糖代谢相关酶的关系[J].果树学报,2014,31(2):250-257.
[22]李明.苹果果实中苹果酸代谢关键酶基因的克隆和表达分析[D].泰安:山东农业大学,2007.