中国农业科学 ›› 2019, Vol. 52 ›› Issue (23): 4374-4385.doi: 10.3864/j.issn.0578-1752.2019.23.017

• 专题:苹果分子生物学 • 上一篇    下一篇

苹果乙烯响应因子MdERF72对非生物胁迫的响应

王佳慧,顾凯迪,王楚堃,由春香,胡大刚(),郝玉金()   

  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,山东泰安 271018
  • 收稿日期:2019-03-21 接受日期:2019-07-01 出版日期:2019-12-01 发布日期:2019-12-01
  • 通讯作者: 胡大刚,郝玉金
  • 作者简介:王佳慧,E-mail:wangjhedu@126.com
  • 基金资助:
    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)

Analysis of Apple Ethylene Response Factor MdERF72 to Abiotic Stresses

WANG JiaHui,GU KaiDi,WANG ChuKun,YOU ChunXiang,HU DaGang(),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:2019-03-21 Accepted:2019-07-01 Online:2019-12-01 Published:2019-12-01
  • Contact: DaGang HU,YuJin HAO

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是乙烯正调控转录因子,在苹果的各组织中均有表达,在果实和茎中表达量相对较高;并且在果实中随着果实的成熟,表达量逐渐升高。苹果组培苗中MdERF72的表达明显受到高盐和低温的诱导。过量表达MdERF72的苹果愈伤组织在高盐和低温胁迫处理下,生长势明显比野生型对照强,电导率,丙二醛、过氧化氢、超氧阴离子的含量都低于野生型,表明MdERF72提高了对盐和低温胁迫的抗性。【结论】MdERF72在响应高盐、低温胁迫过程中发挥着重要的正调控作用,过表达MdERF72可以提高苹果愈伤组织对高盐和低温胁迫的抗性。

关键词: 苹果, MdERF72, 乙烯响应因子, 胁迫应答

Abstract:

【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 Arabidopsis AtERF72, and belonged to the B2 subfamily of ERF family. Analysis of amino acid physicochemical properties indicated that MdERF72 encodes 253 amino acids, and its protein molecular weight was predicted as 27.61 kD, the isoelectric point (pI) was 5.10. In addition, the pro-hydrophobic prediction showed that the hydrophobic portion of MdERF72 was larger than the hydrophilic portion, indicating that it belonged to a hydrophobic protein. Phosphorylation site analysis revealed that MdERF72 had only one threonine phosphorylation site, suggesting that the protein might be regulated by phosphorylation. The results revealed that the MdERF72 promoter sequence contains cis-acting elements associated with jasmonic acid (JA), auxin and drought signals. qRT-PCR analysis showed that MdERF72 was a positive regulatory transcription factor of ethylene, which was expressed in all tissues of apple. Its expression in fruits and stems was relatively high, and gradually increased with the fruit ripening. The expression of MdERF72 in apple tissue culture seedlings was significantly induced by high salt and low temperature. Under the treatment of high salt and low temperature stresses, the MdERF72- overexpressing apple calli had stronger growth potential than the wild type control, and the conductivity, malondialdehyde, hydrogen peroxide and superoxide anion content were lower than the wild type control, indicating that MdERF72 increased the resistance to salt and low temperature stresses.【Conclusion】MdERF72 played an important role in the regulation of high salt and low temperature stresses. Overexpression of MdERF72 could increase the resistance of apple calli to high salt and low temperature stresses.

Key words: apple, MdERF72, ethylene response factor, stress responses