高级检索+

过表达VHA-c4和VHA-c5基因对拟南芥根长的影响

苏杰, 郭荣起, 李国婧, 王瑞刚

苏杰, 郭荣起, 李国婧, 王瑞刚. 过表达VHA-c4和VHA-c5基因对拟南芥根长的影响[J]. 植物科学学报, 2016, 34(5): 765-774. DOI: 10.11913/PSJ.2095-0837.2016.50765
引用本文: 苏杰, 郭荣起, 李国婧, 王瑞刚. 过表达VHA-c4和VHA-c5基因对拟南芥根长的影响[J]. 植物科学学报, 2016, 34(5): 765-774. DOI: 10.11913/PSJ.2095-0837.2016.50765
SU Jie, GUO Rong-Qi, LI Guo-Jing, WANG Rui-Gang. Effect on Root Length of the Overexpression of Arabidopsis VHA-c4 and VHA-c5 Genes[J]. Plant Science Journal, 2016, 34(5): 765-774. DOI: 10.11913/PSJ.2095-0837.2016.50765
Citation: SU Jie, GUO Rong-Qi, LI Guo-Jing, WANG Rui-Gang. Effect on Root Length of the Overexpression of Arabidopsis VHA-c4 and VHA-c5 Genes[J]. Plant Science Journal, 2016, 34(5): 765-774. DOI: 10.11913/PSJ.2095-0837.2016.50765

过表达VHA-c4和VHA-c5基因对拟南芥根长的影响

基金项目: 国家自然科学基金项目(30460018);内蒙古自治区科技创新团队项目(201503004)。
详细信息
    作者简介:

    苏杰(1983-),女,硕士,讲师,研究方向为植物生物化学与分子生物学(E-mail:sujie_1546@163.com)。

    通讯作者:

    王瑞刚(E-mail:ruigangwang@126.com)。

  • 中图分类号: Q943.2

Effect on Root Length of the Overexpression of Arabidopsis VHA-c4 and VHA-c5 Genes

Funds: This work was supported by grants from the National Natural Science Foundation of China (30460018) and the Technology Innovation Team of Inner Mongolia Autonomous Region (201503004).
  • 摘要: 为研究液泡H+-ATPase c亚基VHA-c4和VHA-c5基因在植物生长发育过程中的作用,本研究构建了拟南芥VHA-c4和VHA-c5过表达载体并转化野生型拟南芥,分别获得9个和7个T2代转基因纯合体株系。采用半定量RT-PCR方法对过表达VHA-c4和VHA-c5的转基因纯合体进行阳性鉴定,发现其mRNA表达量均高于对照。对转基因纯合体进行暗培养和正常光照培养,结果显示,黑暗条件下,所有VHA-c4转基因株系的主根变短,而在正常光照下,所有VHA-c5转基因株系的主根变短,推测VHA-c4和VHA-c5分别在黑暗和光照条件下影响植物根的生长。用ABA和糖(葡萄糖和蔗糖)处理转基因纯合体,结果显示它们与野生型的表型无明显差异,表明VHA-c4和VHA-c5基因的过表达没有影响拟南芥对ABA和糖的响应。
    Abstract: To study the role of vacuole H+-ATPase subunit c genes on plant growth and development, over-expression vectors of VHA-c4 and VHA-c5 from Arabidopsis thaliana were constructed and introduced into wild-type A. thaliana, and 9 and 7 homozygous T2 generations of VHA-c4 and VHA-c5 overexpressed transgenic lines were obtained, respectively. Semi-quantitative RT-PCR methods were used for identification of the positive VHA-c4 and VHA-c5 transgenic homozygotes, and their corresponding transcript levels were increased in every transgenic line. When cultivated under different light conditions, the VHA-c4 transgenic plants exhibited a reduction in primary root length compared with that of the wild-type when cultivated in the dark, whereas the VHA-c5 transgenic plants showed reduced primary root length under normal light conditions. These results indicated that VHA-c4 and VHA-c5 affected the growth of roots during light/dark cycles. Following ABA and sugar (glucose and sucrose) treatment, no significant differences were found between the transgenic homozygote phenotype and wild type, indicating that VHA-c4 and VHA-c5 overexpression did not affect the response of Arabidopsis to ABA or sugar.
  • [1] Forgac M. Vacuolar ATPases:rotary proton pumps in physiology and pathophysiology[J]. Nat Rev Mol Cell Biol, 2007, 8(11):917-929.
    [2] Sze H, Schumacher K, Müller LM, Padmanaban S, Taiz L. A simple nomenclature for a complex proton pump:VHA genes encode the vacuolar H+-ATPase[J]. Trends Plant Sci, 2002, 7:157-161.
    [3] Krebs M, Beyhl D, Gorlich E, Al-Rasheid KAS, Marten I, Stierhof YD, Hedrich R, Schumacher K. Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation[J]. Proc Natl Acad Sci USA, 2010, 107:3251-3256.
    [4] Herman EM, Li X, Su RT, Larsen P, Hsu H, Sze H. Vacuo-lartype H+-ATPases are associated with the endoplasmic reticulum and provacuoles of root tip cells[J]. Plant Phy-siol, 1994, 106:1313-1324.
    [5] Dietz KJ, Tavakoli N, Kluge C, Mimura T, Sharma SS, Harris GC, Chardonnens AN, Golldack D. Significance of the V-type ATPase for the adaptation to stressful growth conditions and its regulation on the molecular and biochemical level[J]. J Exp Bot, 2001, 52:1969-1980.
    [6] Hirata T, Iwamoto-Kihara A, Sun-Wada GH, Okajima T, Wada Y, Futai M. Subunit rotation of vacuolar-type proton pumping ATPase:relative rotation of the G as to C subunit[J]. J Biol Chem, 2003, 278:23714-23719.
    [7] Gogarten JP, Fichmann J, Braun Y, Morgan L, Styles P, Taiz SL, DeLapp K, Taiz L. The use of antisense mRNA to inhibit the tonoplast H+-ATPase in carrot[J]. Plant Cell, 1992, 4:851-864.
    [8] Dietz KJ, Rudloff S, Ageorges A, Eckerskorn C, Fischer K, Arbinger B.Subunit E of the vacuolar H(+)-ATPase of Hordeum vulgare L:cDNA cloning, expression and immunological analysis[J]. Plant J, 1995, 8(4):521-529.
    [9] Schumacher K, Vafeados D, Mccarthy M, Sze H, Wilkins T, Chory J. The Arabidopsis det3 mutant reveals a central role for the vacuolar H+-ATPase in plant growth and deve-lopment[J]. Genes Dev, 1999, 13:3259-3270.
    [10] Yoshida S. Chiling-induced inaction and its recovery of tonoplast H-ATPase in mung bean cell supension cultures[J]. Plant Physiol, 1991, 95(2):456-460.
    [11] 毛毅辉, 王天宇, 荣廷昭, 石云素, 宋燕春, 黎裕. 玉米V-ATPase B亚基基因(ZmVHA-B)的克隆及其表达分析[J]. 玉米科学, 2009, 17(2):19-23. Mao YH, Wang TY, Rong TZ, Shi YS, Song YC, Li Y. Cloning and expressional analysis of V-ATPase B subunit gene (ZmVHA-B) on Maize[J]. Journal of Maize Sciences, 2009, 17(2):19-23.
    [12] Narasimhan ML, Binzel ML, Perez-Prat E, Chen Z, Nelson DE, Singh NK, Bressan RA, Hasegawa PM. NaCl regulation of tonoplast ATPase 70-Kilodalton subunit mRNA in tobacco cells. Plant Physiol, 1991, 97(2):562-568.
    [13] Kluge C, Lahr J, Hanitzsch M, Bolte S, Golldack D, Dietz KJ. New insight into the structure and regulation of the plant vacuolar H+-ATPase[J]. J Bioenerg Biomembr, 2003, 35(4):377-388.
    [14] Omri D, Felix F, Nelson N. Crystal structure of yeast V-ATPase subunit C reveals its stator function[J]. EMBO Rep, 2004, 5(12):1148-1152.
    [15] Noumi T, Beltran C, Nelson H, Nelson N. Mutational analy-sis of yeast vacuolar H(+)-ATPase[J]. Proc Natl Acad Sci USA, 1991, 88:1938-1942.
    [16] Zhou A, Bu Y, Takano T, Zhang X, Liu S. Conserved V-ATPase c subunit plays a role in plant growth by influencing V-ATPase-dependent endosomal trafficking[J]. Plant Biotechnol J, 2015, 14:271-283.
    [17] Tsiantis MS, Bartholomew DM, Smith JA. Salt regulation of transcript levels for the c subunit of a leaf vacuolar H(+)-ATPase in the halophyte Mesembryanthemum crystallinum[J]. Plant J, 1996, 9(5):729-736.
    [18] Tyagi W, Rajagopal D, Singla-Pareek SL, Reddy MK, Sopory SK. Cloning and regulation of a stress-regulated Pennisetum glaucum vacuolar ATPase c gene and characte-rization of its promoter that is expressed in shoot hairs and floral organs[J]. Plant Cell Physiol, 2005, 46(8):1411-1422.
    [19] Baisakh N, RamanaRao MV, Rajasekaran K, Subudhi P, Janda J, Galbraith D, Vanier C, Pereira A. Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1(SaVHAc1) gene from the halophyte grass Spartina alterniflora L isel[J]. Plant Biotechnol J, 2012, 10(4):453-464.
    [20] Feng L, Ding H, Wang J, Wang M, Xia W, Zang S, Sheng L. Molecular cloning and expression analysis of RrNHX1 and RrVHA-c genes related to salt tolerance in wild Rosa rugosa[J]. Saudi J Biol Sci, 201522(4):417-423.
    [21] Perera IY, Li X, Sze H. Several distinct genes encode nearly identical to 16 kDa pmtenlipids of the vacuolar H+-ATPase from Arabidopsia thaliana[J]. Plant Mol Biol, 1995, 29:227-244.
    [22] 王瑞刚. 植物启动子的筛选, 调控及外源蛋白的表达调控研究[D].上海:华东师范大学, 2002:1-27. Wang RG. Search for new promoters for the expression of exogenous genes in plants[D]. Shanghai:East China Normal University, 2002:1-27.
    [23] Padmanaban S, Lin X, Perera I, Kawamura Y, Sze H. Differential expression of vacuolar H+-ATPase subunit c genes in tissues active in membrane trafficking and their roles in plant growth as revealed by RNAi[J]. Plant Phy-siol, 2004, 134:1514-1526.
    [24] 徐萍, 李小方, 曾卫军, 王水平. 拟南芥VHA-c3基因的特异性表达和调节[J]. 华东师范大学学报:自然科学版, 2006(2):98-104. Xu P, Li XF, Zeng WJ, Wang SP. Regulation and tissue specific expression of VHA-c3of Arabidopsis Thaliana[J]. Journal of East China Normal University:Natural Science, 2006(2):98-104.
    [25] 郭荣起. 利用RNAi技术对拟南芥液泡H+-ATPase c亚基功能的初步研究[D]. 呼和浩特:内蒙古农业大学, 2008:35-37. Guo RQ. Primary study on the function of subunit c genes of vacuolar H+-ATPase in Arabidopsis thaliana by RNAi[D]. Hohhot:Inner Mongolia Agricultural University, 2008:35-37.
    [26] 于秀敏, 武燕, 王瑞刚. 温度和光照对拟南芥VHA-c基因启动子活性的调节[J]. 河南师范大学学报:自然科学版, 2013, 41(4):120-123. Yu XM, Wu Y, Wang RG. Temperature and light regulates the promoter activity of VHA-c genes of Arabidopsis taliana[J]. Journal of Henan Normal University:Natural Science Edition, 2013, 41(4):120-123.
    [27] 苏杰. 利用过表达对拟南芥液泡H+-ATPase c亚基功能的初步研究[D].呼和浩特:内蒙古农业大学, 2009:20-33. Su J. Primary study on the function of subunit c genes of vacuolar H+-ATPase in Arabidopsis thaliana by overexpression[D]. Hohhot:Inner Mongolia Agricultural University, 2009:20-33.
    [28] 苏杰, 郭荣起, 姜树原, 邸娜, 李国婧, 王瑞刚. 过表达VHA-c1基因对拟南芥根长及ABA与糖响应的影响[J]. 西北植物学报, 2015, 35(5):865-871. Su J, Guo RQ, Jiang SY, Di N, Li GJ, Wang RG. Effect on root length, responses to ABA and sugar by overexpressing Arabidopsis VHA-c1[J]. Acta Botanica Boreali-Occidentalia Sinica, 2015, 35(5):865-871.
    [29] 苏杰, 郭荣起, 李国婧, 王瑞刚. 过表达VHA-c3基因拟南芥对黑暗、ABA与糖的响应[J], 生物技术通报,2016, 32(6):89-95. Su J, Guo RQ, Li GJ, Wang RG. Responses of oerexpressed Arabidopsis VHA-c3 to dark, ABA and sugar[J]. Biotechnology Bulletin, 2016, 32(6):89-95.
    [30] Murray MG, Thompson WF. Rapid isolation of high mole-cular weight plant DNA[J]. Nucleic Acids Res, 1980, 8(19):4321-4325.
    [31] Mersereau M, Pazour GJ, Das A. Efficient transformation of Agrobacterium tumefaciens by electroporation[J]. Gene, 1990, 90(1):149-151.
    [32] Clough SJ, Bent AF. Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J]. Plant J, 1998, 16(6):735-743.
    [33] Huss M, Ingenhorst G, Konig S, Gassel M, Drose S, Zeeck A, Altendorf K, Wieczorek H. Concanamycin A, the specific inhibitor of V-ATPases, binds to the V(0) subunit c[J]. J Biol Chem, 2002, 277(43):40544-40548.
    [34] Smart LB, Vojdani F, Maeshima M, Wilkins TA. Genes involved in osmoregulation during turgor-driven cell expansion of developing cotton fibers are differentially regulated[J]. Plant Physiol, 1998, 116(4):1539-1549.
    [35] Brüx A, Liu TY, Krebs M, Stierhof YD, Lohmann JU, Miersch O, Wasternack C, Schumacher K. Reduced V-ATPase activity in the trans-Golgi network causes oxylipin-dependent hypocotyl growth inhibition in Arabidopsis[J]. Plant Cell, 2008, 20(4):1088-1100.
计量
  • 文章访问数:  1048
  • HTML全文浏览量:  1
  • PDF下载量:  1298
  • 被引次数: 0
出版历程
  • 收稿日期:  2016-03-23
  • 修回日期:  2016-05-09
  • 发布日期:  2016-10-27

目录

    /

    返回文章
    返回