苹果异三聚体G蛋白α亚基基因MdGPA1的克隆及功能鉴定

doi:10.3864/j.issn.0578-1752.2017.03.012

Abstract

Abstract:

【Objective】As an important signal transduction molecule in plant biology, heterotrimeric G protein plays an important role in the stimulation of the external environment, the functions in regulation of responses to biotic and abiotic stresses and transmembrane signal transduction. A α-subunit of the apple heterotrimeric G protein named MdGPA1 was cloned from Malus ×domestica ‘Royal Gala’. Its basic biological functions were identified in transgenic tobacco. It provides a reference for the study of the molecular mechanism of perennial woody plants in response to environmental factor signal transduction. 【Method】MdGPA1 gene was cloned by homology sequence alignment and PCR technique. The phylogenetic tree of GPA1 homologous species was constructed using MEGA5.0. The induced expression and tissue-specific expression profiles of MdGPA1 gene in apple with abiotic stress were detected by real-time fluorescent quantitative PCR (qRT-PCR). A plant over-expression vector of MdGPA1 was constructed and used to transform tobacco by Agrobacterium-mediated method. The phenotypic and physiological performance of the transformants were characterized under drought stresses to investigate the function of MdGPA1 on stress resistance in tobacco.【Result】A α-subunit of the apple heterotrimeric G protein named MdGPA1 (MDP0000309677) was cloned from Malus ×domestic ‘Royal Gala’. Sequence analysis showed that the length of MdGPA1 gene is 1 173 bp, which encoded 390 amino acids. A phylogenetic tree indicated that the apple MdGPA1 exhibited the highest sequence similarity to Pyrus bretschneideri PbGPA1. The qRT-PCR analysis indicated that MdGPA1 has the highest expression levels of expression in apple leaves and response to drought stress, low temperature and salt abiotic stress. Expression levels were significantly down regulated at 150 mmol·L^-1NaCl, 150 mmol·L^-1 mannitol, 10% PEG and 4℃ abiotic stress, and the expression level was significantly increased under 5% H₂O₂ stress treatment. The MdGPA1 transgenic tobacco showed a drought sensitive phenotype. The fresh weight, chlorophyll content of the leaves and proline content of MdGPA1 transgenic tobacco were significantly lower than that of the wild type tobacco. Compared to the wild type, the root morphology of MdGPA1 transgenic tobacco was lower than that of the wild type, and the dry weight was significantly lower than that of the wild type.【Conclusion】The MdGPA1 is involved in the process of plant environment stimulation, and has different responses to abiotic stresses such as drought, low temperature and salt. Transgenic tobacco was more sensitive to drought stress than the wild type tobacco. The experimental results indicate that MdGPA1 plays a negative role in response to drought stress in plants.

Key words: apple, heterotrimeric G protein, MdGPA1, expression analysis, functional identification

LI Rui, AN JianPing, YOU ChunXiang, WANG XiaoFei, HAO YuJin. Molecular Cloning and Functional Characterization of the α-Subunit of Heterotrimeric G Protein Gene MdGPA1 of Apple[J].Scientia Agricultura Sinica, 2017, 50(3): 537-544.

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References

[1] 朱莺, 黄继荣. 植物异三聚体G蛋白研究进展. 植物生理学通讯, 2010(4): 309-316.

Zhu Y, Huang J R. Research progress in plan of heterotrimeric G protein. Chinese Plant Physiology Communications, 2010(4): 309-316. (in Chinese)

[2] Assmann S M. Heterotrimeric and unconventional GTP binding proteins in plant cell signaling. The Plant Cell, 2002, 14(suppl. 1): S355-S373.

[3] Ma H, Yanofsky M F, Meyerowitz E M. Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the USA, 1990, 87(10): 3821-3825.

[4] Jones J C, Duffy J W, Machius M, TEMPLE B R, DOHLMAN H G, JONES A M.. The crystal structure of a self-activating G protein α subunit reveals its distinct mechanism of signal initiation. Science Signaling, 2011, 4(159): ra8.

[5] Millner P A. Heterotrimeric G‐proteins in plant cell signaling. New Phytologist, 2001, 151(1): 165-174.

[6] Wang X Q, Ullah H, JONES A M, ASSMANN S M. G protein regulation of ion channels and abscisic acid signaling in Arabidopsis guard cells. Science, 2001, 292(5524): 2070-2072.

[7] Pandey S, Assmann S M. The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein α subunit GPA1 and regulates abscisic acid signaling. The Plant Cell, 2004, 16(6): 1616-1632.

[8] CHAKRAVORTY D, TRUSOV Y, ZHANG W, ACHARYA B R, SHEAHAN M B, MCCURDY D W, ASSMANN S M, BOTELLA J R. An atypical heterotrimeric G-protein γ-subunit is involved in guard cell K⁺-channel regulation and morphological development in Arabidopsis thaliana. The Plant Journal, 2011, 67(5): 840-851.

[9] 马鲜歌,贺军民. 异三聚体G蛋白在UV-B诱导拟南芥气孔关闭中

的作用. 中国农业科学, 2012, 45(5): 848-853.

MA X G, HE J M. Role of heterotrimeric G protein in UV-B-Induced Arabidopsis stomatal closure. Scientia Agricultura Sinica,2012, 45 (5):848-853. (in Chinese)

[10] Jones A M, Ecker J R, Chen J G. A reevaluation of the role of the heterotrimeric G protein in coupling light responses in Arabidopsis. Plant Physiology, 2003, 131(4): 1623-1627.

[11] Ullah H, Chen J G, Young J C, IM K H, SUSSMAN M R, JONES A M. Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis. Science, 2001, 292(5524): 2066-2069.

[12] Perfus-Barbeoch L, Jones A M, Assmann S M. Plant heterotrimeric G protein function: insights from Arabidopsis and rice mutants. Current Opinion in Plant Biology, 2004, 7(6): 719-731.

[13] Temple B R S, Jones A M. The plant heterotrimeric G-protein complex. Annu. Rev. Plant Biol., 2007, 58: 249-266.

[14] Huang H, Weiss C A, Ma H. Regulated expression of the Arabidopsis G protein α subunit gene GPA1. International Journal of Plant Sciences, 1994, 155(Volume 155, Number 1): 3-14.

[15] GAZZARRINI S, MCCOURT P. Cross-talk in plant hormone signalling: what arabidopsis mutants are telling us. Annals of Botany, 2003, 91(6): 605-612.

[16] Weerasinghe R R, Swanson S J, Okada S F, GARRETT M B, KIM S Y, STACEY G, BOUCHER R C, GILROY S, JONES A M. Touch induces ATP release in Arabidopsis roots that is modulated by the heterotrimeric G-protein complex. FEBS Letters, 2009, 583(15): 2521-2526.

[17] Coursol S, Fan L M, Le Stunff H, SPIEGEL S, GILROY S, ASSMANN S M. Sphingolipid signalling in Arabidopsis guard cells involves heterotrimeric G proteins. Nature, 2003, 423(6940): 651-654.

[18] Trusov Y, Jordá L, Molina A, BOTELLA J R. G Proteins and Plant Innate Immunity. Integrated G Proteins Signaling in Plants. Springer Berlin Heidelberg, 2010: 221-250.

[19] Okamoto H, Matsui M, Deng X W. Overexpression of the heterotrimeric G-protein α-subunit enhances phytochrome-mediated inhibition of hypocotyl elongation in Arabidopsis. The Plant Cell, 2001, 13(7): 1639-1652.

[20] CHEN J G, GAO Y, JONES A M. Differential roles of Arabidopsis heterotrimeric G-protein subunits in modulating cell division in roots. Plant Physiology, 2006, 141(3):887-897.

[21] Lease K A, Wen J, Li J, DOKE J T, LISCUM E, WALKER J C. A mutant Arabidopsis heterotrimeric G-protein β subunit affects leaf, flower, and fruit development. The Plant Cell, 2001, 13(12): 2631-2641.

[22] Wu Y, Xu X, Li S, LIU T, MA L G, SHANG Z L. Heterotrimeric G‐protein participation in Arabidopsis pollen germination through modulation of a plasmamembrane hyperpolarization-activated Ca²⁺- permeable channel. New Phytologist, 2007, 176(3): 550-559.

[23] CHAKRAVORTY D, BOTELLA J R. Over-expression of a truncated Arabidopsis thaliana, heterotrimeric G protein γ subunit results in a phenotype similar to α and β subunit knockouts. Gene, 2007, 393(1–2):163-170.

[24] Ullah H, Chen J G, Temple B, BOYES D C, ALONSO J M, DAVIS K R, ECKER J R, JONES A M. The β-subunit of the Arabidopsis G protein negatively regulates auxin-induced cell division and affects multiple developmental processes. The Plant Cell, 2003, 15(2): 393-409.

[25] Choudhury S R, Pandey S. Specific subunits of heterotrimeric G proteins play important roles during nodulation in soybean. Plant Physiology, 2013, 162(1): 522-533.

[26] Schuler B, Seckler R.. Role of a heterotrimeric G protein in regulation of Arabidopsis seed germination. Plant Physiology, 2002, 129(2): 897-907.

[27] WARPEHA K M, UPADHYAY S, YEH J, ADAMIAK J, HAWKINS S I, LAPIK Y R, ANDERSON M B, KAUFMAN L S. The GCR1, GPA1, PRN1, NF-Y signal chain mediates both blue light and abscisic acid responses in Arabidopsis. Plant physiology, 2007, 143(4): 1590-1600.

[28] Zhang L, Wei Q, Wu W, CHENG Y, HU G, HU F, SUN Y, ZHU Y, SAKAMOTO W, HUANG J. Activation of the heterotrimeric G protein α-subunit GPA1 suppresses the ftsh-mediated inhibition of chloroplast development in Arabidopsis. The Plant Journal, 2009, 58(6): 1041-1053.

[29] Nilson S E, Assmann S M. The α-subunit of the Arabidopsis heterotrimeric G protein, GPA1, is a regulator of transpiration efficiency. Plant Physiology, 2010, 152(4): 2067-2077.

[30] CHAKRABORTY N, SHARMA P, KANYUKA K, PATHAK R R, CHOUDHURY D, HOOLEY R, RAGHURAM N. G-protein α-subunit (GPA1) regulates stress, nitrate and phosphate response, flavonoid biosynthesis, fruit/seed development and substantially shares GCR1 regulation in A. thaliana. Plant Molecular Biology, 2015, 89(6): 559-576.

[31] 张占全, 宋水山, 张英春, 杨文香, 刘大群. 异三聚体G蛋白参与小麦抗叶锈病反应的研究. 中国农业科学, 2009, 42(1): 117-123.

ZHANG Z Q, SONG S S, ZHANG Y C, YANG W X, LIU D Q. Involvement of the heterotrimeric G protein in the defense responses of wheat to Puccinia triticina. Scientia Agricultura Sinica, 2009, 42(1): 117-123. (in Chinese)

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Molecular Cloning and Functional Characterization of the α-Subunit of Heterotrimeric G Protein Gene MdGPA1 of Apple

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