ФИЗИОЛОГИЯ РАСТЕНИЙ, 2013, том 60, № 5, с. 754-760
КРАТКИЕ СООБЩЕНИЯ
УДК 581.1
CLONING AND CHARACTERIZATION OF ETHYLENE INSENSITIVE 2 (EIN2)
GENE FROM Cucumis melo1 © 2013 F. Gao*, **, 2, J. Hao*, **, 2, Y. Yio*, **, X. Wang*, A. Hasi*, **
*College of Life Sciences, Inner Mongolia University, Hohhot, P.R. China **Inner Mongolia Key Laboratory of Herbage and Endemic Crop Biotechnology, Hohhot, P.R. China
Received July 26, 2012
Melon is an ideal alternative model fruit to examine ethylene perception and sensitivity. Ethylene insensitive 2 (EIN2), an integral membrane protein in the endoplasmic reticulum, is an important regulator of ethylene and other phytohormone signaling. We isolated a cDNA clone that encoded EIN2 homolog for the first time on the basis of melon (Cucumis melo L. cv. Hetao) fruit total RNA by in silico cloning and reverse-transcription PCR (RT-PCR). The cDNA contained an open reading frame of 3876 bp corresponding to a polypeptide of 1291 amino acids with a predicted mol wt of141 kD. The expression patterns of different developmental stages of fruit, vegetative organs, and reproductive tissues and upon the treatment with IAA and ABA were analyzed. CmEIN2 mediates ethylene signals in many processes and is a component of signal transduction by ethylene, auxin, and abscisic acid.
Keywords: Cucumis melo — EIN2 — ethylene — gene expression
DOI: 10.7868/S0015330313050047
INTRODUCTION
The plant hormone ethylene triggers a wide range of physiological and morphological responses in plant, including the inhibition of cell expansion, the promotion of leaf and flower senescence, the induction of fruit ripening and abscission, the resistance to pathogen and insect attack, and the adaptation to stress conditions [1]. Many components in the ethylene signaling pathway have been identified, using genetic mutants of the model plant Arabidopsis thaliana, and a largely linear pathway in its early steps has been established and expanded into an increasingly complex signaling system [2].
Ethylene insensitive 2 (EIN2), an integral membrane protein in the endoplasmic reticulum [3], is a central regulator of the ethylene signaling pathway and its gene is the only one in this pathway whose loss-of-function mutations effect complete insensitivity to ethylene [4]. Genetic data indicate that EIN2 mediates an essential step in ethylene signaling between
1 This text was submitted by the authors in English.
2 These authors contributed equally to this work.
Abbreviations: CTR1 - constitutive triple responsel; DAP - days after pollination; EBF - EIN3-binding F-box; EIL - ethylene-insensitive3-like; EIN - ethylene insensitive; ETP - EIN2-tar-geting protein.
Corresponding authors: A. Hasi. College of Life Sciences, Inner Mongolia University, Hohhot 010021, P.R. China. E-mail: hasind@sina.com
CTR1 and EIN3/EIL [5]. Recent studies have demonstrated that ethylene-induced stabilization of EIN3 is mediated by proteasomal degradation of EBF1 and EBF2, which requires EIN2 [6], and that the degradation of EIN2 is triggered by the ethylene-controlled F-box proteins ETP1 and ETP2 [7]. However, few EIN2 genes have been isolated from plants, such as Arabidopsis thaliana [4], Petunia x hybrida [8], Oryza sativa [9], Lycopersicon esculentum [10], Medicago truncatula [11], Prunus persica [12], and Dianthus caryophyllus [13], and little is known about the function of EIN2 in species other than Arabidopsis.
One of the most frequently studied examples of ethylene regulation is the ripening of climacteric fruit. Melon is an ideal, alternative model fruit to examine ethylene perception and sensitivity with regard to fruit ripening, because the ethylene-dependent and -independent regulatory pathways coordinate ripening in melon fruit [14, 15]. Based on the whole-genome shotgun sequence of the cucumber, an important cultivated plant of Cucubitaceae, the mean sequence similarity over coding regions between cucumber and melon is 95% [16]. This information provided the additional genomic approach to learn melon. To increase our understanding of the function of EIN2 in ethylene signaling and responses, we isolated a homolog of Arabidopsis EIN2 from melon by in silico cloning and RT-PCR and analyzed its expression patterns.
MATERIALS AND METHODS
Plant material and hormonal treatments. Melon (Cucumis melo L. cv. Hetao) plants were grown on a farm. Self-pollination was performed manually, and the pollination time was recorded and controlled between 9:00 and 11:00 a.m. Only one fruit was remained on each plant. Fruits were harvested at 9:0010:00 a.m. every 5 days from 15 to 30 DAP and daily from 31 DAP until rotting, based on the DAP and maturity indices. Other tissues, such as roots, stems, young leaves, petals, and ovaries, were also collected from the greenhouse.
To minimize the effects of endogenous hormones (ethylene, auxin, and ABA) on CmEIN2 transcription, sterile young leaves were transferred to 250-mL flasks that contained 100 mL of 0.5 MS liquid medium with the appropriate treatment. The flasks were incubated on a rotary shaker (100 rpm) for 2 h at 30°C. The treatments with IAA and ABA (at 0.4, 4, and 40 ^M) were performed. Leaves that were kept in basal medium without any hormone were used as the control. At the end of the treatment, all leaves were removed and briefly blotted dry.
All plant materials were frozen immediately in liquid nitrogen and stored at -80°C for RNA analysis.
Internal ethylene measurements. The endogenous gas sample was collected with a syringe from the fruit cavity at 15, 20, 25, and 30 DAP and daily from 31 DAP until rotting and measured by gas chromatography (GC-9A, "Shimadzu", Japan) to determine the internal ethylene production.
Extraction of total RNA. Total RNA from meso-carp tissue was isolated as described [17]. For other tissues, total RNA was extracted using RNAiso for polysaccharide-rich plant tissue ("Takara", Japan) according to the manufacturer's instructions. All RNA extracts were analyzed by agarose gel electrophoresis and UV spectrophotometry.
Cloning and in silico analysis of melon CmEIN2 cDNA.
Mesocarp RNA was used to clone full-length melon cDNA homologs of Arabidopsis EIN2 by RT-PCR. First-strand cDNA synthesis was performed using the ThermoScript™ RT-PCR System ("Invitrogen", United States) according to the manufacturer's instructions.
Degenerate gene-specific primers were designed as follows. (1) In silico analysis was used to extend the melon CmEIN2 sequence in the Cucurbit Genomics Database (http://www.icugi.org/), based on the sequence of CmEIN2 EST (GenBank ID: AM717510) [18]. (2) The extended CmEIN2 sequence was searched against the NCBI whole-genome shotgun contigs (wgs) database using BLASTN (http:// www.ncbi.nlm.nih.gov/blast/Blast.cgi), optimizing for discontiguous megablast, to obtain cucumber con-tigs that contain the genomic DNA sequence of the putative CsEIN2. (3) The gene prediction program
Softberry of FGENESH (http://linux1.softberry.com/ berry.phtml) was used to predict multiple (alternative splice) variants of potential CsEIN2 genes in the cucumber genome contig to identify the initiation and termination codons of the putative CsEIN2.
Using first-strand cDNA as a template, the predicted CmEIN2 cDNA was amplified with the degenerate primers 5' - GATGGAATCTACGACATTG-CATAC-3' (forward) and 5'-ACTATGAGC-TATAAGGAACGGATG-3' (reverse), using PrimeSTAR™ HS DNA polymerase ("Takara"). The PCR product was purified, ligated into pMD19-T ("Takara"), transformed into Escherichia coli JM109, and sequenced. Full-length amino acid alignments were performed using DNAMAN ("Lynnon Biosoft", United States). The phylogenetic analysis was conducted with ClustalX (2.0.12) (ftp://ftp.ebi.ac.uk/pub/software /clustalw2/2.0.12/). The phylogenetic tree was constructed using MEGA v. 5.
Relative quantitative real-time RT-PCR. CmEIN2 mRNA levels were measured by quantitative PCR. Primers were designed with a calculated Tm of 60~65°C, and the amplification product was 155 bp: forward primer 5'-ATGGTCAAGGATGTGGAGATAGC-3'; reverse primer 5'-TCGTGAGTGGCAACTGGTTTA-3'. For each trial, 6 independent experiments were used as three biological replicates and two records, and all relative fold-differences in expression were normalized to GAPDH (GenBank ID: AB033600) (forward primer 5' - ATCATTCCTAGCAGCACTGG- 3' and reverse primer 5'-TTGGCATCAAATATGCTTGACCTG-3').
First-strand cDNA was synthesized using the Pri-meScript® RT Reagent Kit (Perfect Real Time) ("Takara") according the manufacturer's protocol. For the cDNA synthesis, 0.5 ^g of total RNA from melon fruit at various stages of development and ripening and other tissues was used as a template in a 10-^L of the reaction mixture. SYBR® Premix ex TaqTM (Perfect Real Time) ("Takara") was used for the real-time RT-PCR, including 5 ^M of each primer, and the reactions were run on an Opticon 3 Real-Time PCR System ("BioRad", United States). Melting curves were generated immediately after the last cycle to exclude any influence of primer dimers. Cycle numbers, at which the fluorescence passed the cycle threshold (Ct), were analyzed, and the relative expression was calculated by 2(-AACt)-method.
RESULTS
Cloning of CmEIN2 and sequence analysis
The melon CmEIN2 sequence could be only extended to 1255 bp (matched with melon unigene MU49291, Database: melon_unigene_v. 4) in the Cucurbit Genomics Database. Thus, it was necessary to search for cucumber whole-genome shotgun contigs that harbored CsEIN2. The BLAST results matched with cucumber contig 6472 (GenBank ID:
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