苹果乙烯响应因子MdERF72对非生物胁迫的响应  

Analysis of Apple Ethylene Response Factor MdERF72 to Abiotic Stresses

作　　者：王佳慧 顾凯迪 王楚堃 由春香[1] 胡大刚[1] 郝玉金[1] WANG JiaHui;GU KaiDi;WANG ChuKun;YOU ChunXiang;HU DaGang;HAO YuJin(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)

机构地区：[1]山东农业大学园艺科学与工程学院/作物生物学国家重点实验室/农业部黄淮地区园艺作物生物学与种质创制重点实验室

出　　处：《中国农业科学》2019年第23期4374-4385,共12页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31601728,31430074,31772288);山东省现代农业产业技术体系(SDAIT-06-03);国家现代苹果产业技术体系(CARS-27);山东省自然科学基金(ZR2016CQ13)

摘　　要：【目的】乙烯响应因子(ethylene response factor,ERF)是植物特有的一类转录因子,参与植物根系形成、下胚轴伸长、果实成熟、器官衰老等生长发育过程,在调节植物生物和非生物胁迫反应及果实品质过程中发挥着至关重要的作用。克隆苹果乙烯响应因子MdERF72,通过表达分析和转基因功能分析,研究其在抵御非生物胁迫过程中的功能,为探索MdERF72在植物生长发育过程中的功能提供理论依据。【方法】以‘王林’苹果(Malus×domestica Borkh.)愈伤组织为试材,利用RT-PCR技术克隆MdERF72,利用生物信息学方法分析其编码氨基酸序列组成、蛋白质理化性质、亲缘关系、空间结构等,并利用MEGA5.0构建系统进化树,与拟南芥ERF-B2亚家族进行蛋白序列同源性分析;利用实时荧光定量PCR技术探明其在苹果组织中的表达和果实发育时期的时空表达特征;同时利用实时荧光定量PCR技术检测‘嘎拉’苹果组培苗中MdERF72对ACC、NaCl以及低温的响应;构建基因的过表达载体,通过农杆菌介导的遗传转化获得稳定遗传的过表达苹果愈伤组织;检测NaCl以及低温处理后,野生型和转基因苹果愈伤组织的鲜重、丙二醛含量、电导率、过氧化氢含量以及超氧阴离子含量的差异。【结果】MdERF72位于苹果第13号染色体上,该基因存在1个ERF家族特有的AP2/ERF结构域。进化树分析结果显示,MdERF72与拟南芥AtERF72序列同源性较高,都属于ERFs家族的B2亚家族。氨基酸理化性质分析表明,MdERF72编码253个氨基酸,预测其蛋白质分子量为27.61 kD,等电点(pI)为5.10。另外,亲疏水预测结果显示MdERF72疏水部分大于亲水部分,表明其属于疏水性蛋白。磷酸化位点分析显示,MdERF72只有苏氨酸磷酸化位点,表明该蛋白可能受到磷酸化作用的调控。MdERF72启动子序列中含有与茉莉酸(JA)、生长素及干旱信号相关的顺式作用元件。MdERF72是乙烯正调控【Objective】Ethylene response factor(ERF), a plant-specific transcription factor, is involved in the growth and development of root formation, hypocotyl elongation, fruit ripening, and organ senescence. It also plays a vital role in regulating responses of plant biological and abiotic stress, as well as fruit qualities. In this study, we cloned the apple ethylene response factor MdERF72. Subsequently, a series of expression analysis and functional identification of transgenic apple calli were performed to study its role in abiotic stress responses. These results provided a theoretical basis for exploring the functions of MdERF72 in plant growth and development.【Method】Using Orin apple calli(Malus calli stica Borkh.) as the test material, the MdERF72 was cloned from apple fruits by RT-PCR assay. Bioinformatics methods were used to analyze its amino acid sequence, physicochemical properties genetic relationship, and spatial structure. MEGA5.0 was used to construct the phylogenetic tree for analyzing the homology of its protein sequence with ERF-B2 subfamily in Arabidopsis. The real-time fluorescence PCR(qRT-PCR) assays were performed to analyse the expression of MdERF72 in different organs and tissues of apple, as well as in apple fruits during different developmental stages. Meanwhile, the expression of MdERF72 in Gala apple tissue culture seedlings treated with ACC, NaCl and low-temperature was detected by qRT-PCR assay. We also constructed its overexpression vector and obtained stable overexpression apple calli through Agrobacterium-mediated genetic transformation. The fresh weight, malondialdehyde content, electrical conductivity, hydrogen peroxide content and superoxide anion content of the wild type and transgenic apple calli were detected after NaCl and low temperature treatment. 【Result】 MdERF72 was located on chromosome 13 in apple genome, which had an AP2/ERF domain, unique to ERF family. Phylogenetic tree analyses indicated that the apple MdERF72 exhibited the highest sequence similarity to Ar

关 键 词：苹果 MdERF72 乙烯响应因子 胁迫应答 

分 类 号：S66[农业科学—果树学]