Expression analysis and preliminary functional characterization of the apple MdRCD1

Abstract: 【Objective】Plants are constantly exposed to a broad spectrum of biotic and abiotic stresses during growth and development in nature, such as salt, drought, cold and oxidation.In order to adapt to the environment, plants have developed versatile stress-response mechanisms.Recent studies have shown that SROs (SIMILAR-TO-RCD-ONE) play important roles in response to stresses in diverse species.SROs have been identified as positive regulators to protect plants from oxidation and salt damage.However, the function of SROs in woody plants remains largely unknown, particularly in apples.In this study, we isolated and cloned a SRO gene, named as MdRCD1.Furthermore, we analyzed the protein structure and expression patterns of MdRCD1, and researched the functions of MdRCD1 in apple callus.【Methods】The phylogenetic tree of SROs proteins were constructed using a neighbor-joining method associated with the MEGA5 program (http://www.megasoftware.net/) .The image of the phylogenetic tree of SROs proteins was drawn in MEGA5.Homology researches of the NCBI (National Center for Biotechnology Information) databases were performed using BLAST (http://www.ncbi.nlm.nih.gov/BLAST) with default parameters.The full-length c DNA of MdRCD1 was determined by PCR.Tissue culture seedlings of‘Gala’were used to clone MdRCD1 and analyze its expression levels in various stress conditions.Five years of strong‘Gala’trees were used to study the tissue expression pattern of MdRCD1 in different tissues.The tissue culture seedlings were grown on an Murashige and Skoog (MS) medium with 0.1 mg·L^-1of gibberellins, 0.5 mg·L^-1of 6-Benzylaminopurine (6-BA) and 0.2 mg·L^-1 of 1-Naphthaleneacetic acid (NAA) at 25℃, the tissue cultures were subcultured at monthly intervals under the long-day photoperiod (16-h-light/8-h-dark) .The‘Orin’callus was grown on an MS medium containing 1.5 mg·L-12, 4-dichlorophenoxy (2, 4-D) and 0.5 mg·L^-1of 6-BA in the dark at 25℃, and subcultured at 15-20 d intervals.To mimic salt, osmosis, ABA, high temperature-induced stress, the tissue cultures were treated with an MS medium containing 100 mmol·L^-1NaCl, 200 mmol mannitol, 100μmol ABA, 40℃, respectively, then samples were harvested at denoted times and stored at-80℃for further analysis.Gene expression analysis was performed using q RT-PCR.In order to construct the MdRCD1 overexpression vector, its full-length c DNA was first cloned into the PMD18-T vector.After sequencing, the plasmid was digested by Eco RⅠand SalⅠ, and then the recovered fragment was introduced into the PRI-101 expression vector.The recombinant plasmid was transferred into an Agrobacterium tumefaciens strain LBA4404, and then introduced into the apple callus with an Agrobacterium-mediated method.To test the function of MdRCD1 on salt resistance, the wild-type callus and MdRCD1 transgenic callus at the same growth state were treated with different concentrations of salt for 15 d.Then the growth state of the wild-type and MdRCD1 callus were observed.Meanwhile, we measured the fresh weight and analyzed the content of proline and malonaldehyde (MDA) in them.【Results】MdRCD1 (MDP0000234325) was cloned from‘Gala’apple (Malus×domestica‘Gala’) .Sequence analysis indicated that the Open Reading Frame (ORF) of MdRCD1 contained 1 803 bp, encoding a protein of 600 amino acid residues.Its molecular mass was predicated to be 67.39 ku, and p I was 7.06.The results exhibited the homology of the amino acids sequence of MdRCD1 with RCD1s from other species.Multiple sequence alignment and phylogenetic analysis showed that MdRCD1 had the highest evolutionary relationship with Pb RCD1 from the pear, and had a classic WWE domain in the N-terminal.The results of the protein sequence alignment indicated that MdRCD1was highly conserved among SROs family in diverse species.The q RT-PCR results demonstrated that MdRCD1 extensively existed in various apple tissues, and the expression level in the stem was highest, suggesting that MdRCD1 is a constitutive expression gene.In addition, MdRCD1 was induced by multiple stresses including ABA, Na Cl and osmotic.The open reading frames (ORFs) of MRCD1 were introduced into the PRI-101 plant transformation vector downstream of the cauliflower mosaic virus (Ca MV) 35S promoter, and then the recombinant structure was transformed into Agrobacterium tumefaciens LBA4404.Transgenic apple callus was generated by Agrobacterium-mediated genetic transformation.Under normal conditions, the growth of MdRCD1 and wild-type apple callus showed no differences.However, in the condition of salt stress, overexpression of MdRCD1 in apple callus significantly enhanced resistance compared to wild-type callus.In addition, the fresh weight and proline content of MdRCD1 callus were higher than wild-type callus, and the MDA content was lower.These results revealed that MdRCD1 may play an important role in apple response to salt stress.【Conclusion】Taken together, MdRCD1 is a member of the SROs family and the protein sequences of the RCD1 are conserved among different species.MdRCD1 is widely expressed in all tissues tested in apples, including root, stem, flower, leaf and fruit, however its expression levels are different.MdRCD1 is induced by multiple stresses such as Na Cl, ABA and osmotic stresses.Overexpressing MdRCD1 in apple callus can significantly improve salinity tolerance.Therefore, we speculate that MdRCD1 may play an important role in salt stress.MdRCD1 also can be used as a resistance gene to screen stock, resulting in improving the production of apples.