Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (23): 4374-4385.doi: 10.3864/j.issn.0578-1752.2019.23.017

• SPECIAL FOCUS: MOLECULAR BIOLOGY OF APPLE • Previous Articles     Next Articles

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 E-mail:fap_296566@163.com;haoyujin@sdau.edu.cn

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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

Fig. 1

PCR amplification of MdERF72"

Fig. 2

Sequence alignment of MdERF72 in apple and its homologous"

Fig. 3

Phylogenetic tree between apple ERF protein MDP0000756341 and Arabidopsis ERF-B2 family proteins"

Table 1

Some important cis-acting regulatory elements in the promoter of MdERF72"

调控序列
Regulatory sequence		 序列
Sequence		 位点功能
Function of site		 位置
Location
Sp1 GGGCGG 光响应元件Light responsive element +51
TGA-box TGACGTAA 生长素响应元件Part of an auxin-responsive element -960
LTR CCGAAA 低温响应元件cis-acting element involved in low-temperature responsiveness -600
ARE AAACCA 厌氧响应元件cis-acting regulatory element essential for the anaerobic induction +1238
MBS CAACTG 干旱胁迫响应元件MYB binding site involved in drought-inducibility +1192
TGACG-motif TGACG 茉莉酸响应元件cis-acting regulatory element involved in the MeJA-responsiveness +768, -841
CAT-box GCCACT 分生组织表达响应元件cis-acting regulatory element related to meristem expression -1986

Fig. 4

Expression analysis of MdERF72 of Gala apple tissue culture seedlings in response to ACC Different letters indicate significant difference at 0.05 levels. The same as below"

Fig. 5

Expression of MdERF72 gene in different tissues of apple"

Fig. 6

Expression of MdERF72 gene in different developmental stages of apple"

Fig. 7

Expression analysis of MdERF72 of Gala apple tissue culture seedlings in response to NaCl (A) and 4℃ (B) low temperature"

Fig. 8

Identification of MdERF72 transgenic apple calli"

Fig. 9

Growth status of wild type (WT) and MdERF72 overexpressing apple calli (OE1, OE2 and OE3) under NaCl"

Fig. 10

Fresh weight (A), Malondialdehyde content (B), electronic conductivity (C), Hydrogen peroxide content(D) and Superoxide anion content (E) of wild type (WT) and MdERF72 overexpressing apple calli (OE1, OE2 and OE3) under NaCl treatments"

Fig. 11

Growth status of wild type (WT) and MdERF72 overexpressing apple calli (OE1, OE2 and OE3) under 4℃ low temperature"

Fig. 12

Fresh weight (A), Malondialdehyde content (B), electronic conductivity (C), Hydrogen peroxide content (D) and Superoxide anion content (E) of wild type (WT) and MdERF72 overexpressing apple calli (OE1, OE2 and OE3) under 4℃ low temperature"

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