Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (3): 537-544.doi: 10.3864/j.issn.0578-1752.2017.03.012

• HORTICULTURE • Previous Articles     Next Articles

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

LI Rui, AN JianPing, YOU ChunXiang, WANG XiaoFei, HAO YuJin   

  1. College of Horticulture Science and Engineering, Shandong Agricultural University/State Key Laboratory of Crop Biology/MOA Key Laboratory of Horticultural Crop Biology（Huanghuai Region）and Germplasm Innovation, Tai’an 271018, Shandong
  • Received:2016-05-27 Online:2017-02-01 Published:2017-02-01

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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

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