苹果异三聚体G蛋白α亚基基因MdGPA1的克隆及功能鉴定

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

【目的】异三聚体G蛋白(Heterotrimeric G protein)作为植物生物体内重要的信号转导分子,在感受外界环境刺激、参与植物抗逆反应和跨膜信号转导等方面发挥着重要作用。克隆异三聚体G蛋白α亚基基因MdGPA1,并在烟草中过量表达MdGPA1,对其进行生物学功能鉴定和生理指标分析,为多年生木本植物响应环境因子信号转导过程中的分子机理研究提供参考。【方法】本研究以‘嘎拉’苹果(Malus×domestica‘Royal Gala’)为研究试材,利用同源序列比对和PCR技术,克隆获得MdGPA1。使用MEGA5.0构建GPA1物种间系统进化树;利用qRT-PCR方法检测该基因在苹果受非生物胁迫诱导表达及组织特异性表达情况。构建MdGPA1植物过表达载体,通过农杆菌介导法转化烟草叶片,比较干旱胁迫条件下野生型和转基因株系的表型与生理指标,验证MdGPA1在植物干旱胁迫条件下的生物学功能。【结果】克隆得到苹果异三聚体G蛋白α亚基基因MdGPA1(基因序列号:MDP0000881842),该基因长为1 173 bp,编码390个氨基酸。进化树分析表明MdGPA1与白梨Pb GPA1亲缘关系最近,同源性最高。基因表达分析显示MdGPA1主要在叶片中表达,在根系中的表达量次之,在茎和果实中的表达量较低。定量分析表明,该基因参与干旱、低温和盐等非生物逆境胁迫响应,在150 mmol·L^(-1)Na Cl、150 mmol·L^(-1)甘露醇、10%PEG和4℃胁迫条件下表达量明显下调,在5%H_2O_2胁迫处理下表达量明显上调。在烟草中过量表达MdGPA1,发现MdGPA1转基因烟草表现出对干旱敏感的表型特征,其叶片鲜重、叶绿素含量以及脯氨酸含量明显低于野生型烟草。在地下部,MdGPA1转基因烟草同样表现出对干旱敏感的表型特征;其根系形态相比于野生型较小,干重也明显低于野生型。【结论】MdGPA1参与了植物感受外界环境刺激的过程,对干旱、低温和盐等非生物逆境胁迫都存在着不同程度的响应。在烟草中异源表达MdGPA1后,提高了烟草对干旱的敏感性,转基因烟草表现出不耐干旱的表型,受干旱胁迫比野生型烟草更为严重,说明MdGPA1在响应植物抗旱胁迫中起着负调控作用。

[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 ct-subunit of the apple heterotrimeric G protein named MdGPA1 was cloned from Malus xdomestica ＇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% H202 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 MdGPA 1 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.