Cloning and expression analysis of PavMYC2 gene in Prunus avium L.

Abstract:【Objective】Sweet cherry (Prunus avium L.) is an important economic fruit crop in temperate regions of the world. Temperature stress is closely involved in flower bud differentiation. JA-activated transcription factor MYC2, which is the most extensively studied components in the JA signaling pathway, plays a regulatory role in temperature stress. However, the MYC2 gene has been less studied in sweet cherry. In the present research, PavMYC2 gene was cloned successfully, and the functional localization of MYC2 gene was identified. Moreover, its expression pattern during organogenesis, dormancy and flowering stages was analyzed.【Methods】The sweet cherries Royal Lee and Hongdeng were selected for the current experiment, which were grafted on Chinese cherry rootstock (P. pseudocerasus Lindl.‘Daqingye'). Royal Lee is a low chilling requirement cultivar from the breeding program of low-chill sweet cherries in California, USA, and Hongdeng is a high chilling requirement cultivar from China.Both cultivars were grown in the experimental farm at Shanghai Jiao Tong University in Shanghai (121.48°E, 31.25°N). Floral buds of sweet cherries were collected on 15 July, 15 August, 15 September,15 October, 15 November and 15 December in 2019; and 15 January, 5 February, 5 March, 10 March and 15 March in 2020. All materials were collected for three biological replicates. These buds were frozen in liquid nitrogen and stored at -80 ℃ before RNA extraction. According to the manufacturer's instructions, total RNA was extracted using an RNAprep purePlant Kit (TianGen, China). To isolate the full-length cDNA of these genes, 1 µg of total RNA was converted into cDNA using PrimeScriptTMII1st Strand cDNA Synthesis Kit(TaKaRaBiotechnology, Dalian, China) and was subsequently diluted five times with sterile water. Primers were designed using Primer 5 software. The PCR-products were cloned into the p EASY®-Blunt Cloning Vector (TransGen Biotech, Beijing, China), and then sequenced. Phylogenetic and molecular evolutionary analysis were conducted using MEGA version 6. To generate a phylogenetic tree, the complete sequences of the other species were obtained from the GenBank DNA database. The Neighbor-Joining method in MEGA was used to construct different trees. The reliability of the obtained trees was tested using bootstrapping with 1000 replicates. RT-qPCR was performed on a Bio-Rad System(Bio-Rad, CA, USA). The procedure was conducted as follows: 95 ℃ for30 s, amplification for 40 cycles(95 ℃ for 5 s, and 60 ℃ for 30 s). PavMYC2 cDNAs were cloned into PHB vectors containing a cauliflower mosaic virus(CaMV) 35S promoter, a translation enhancer and a GFP fluorescent protein tag in the subcellular localization assessment. We cloned the CDS of PavMYC2and PavJAZ1-6 into the vector pXY104 and pXY106 for BiFC assay, respectively. Constructed vectors were transformed into Agrobacterium tumefaciens strain GV3101 and subsequently cultured to an OD600of approximately 0.8-1.0. The mixed suspension liquid with pairs were co-transformed into five-weekold leaves of Nicotiana benthamiana after 2 to 5 h. Yellow fluorescent protein(YFP) signals were detected after 48-72 h by a laser scanning confocal microscope (ZeissLSM510/ConfoCor2).【Results】PavMYC2 contained an open reading frame(ORF) of 689 bp that encoded a predicted protein of 237 amino acids. It contained a conserved domain bHLH-Zip, belonging to bHLH family. To examine the subcellular localization of PavMYC2 in sweet cherry, the coding sequence of PavMYC2 was fused to the PHBGFP vector. The PHB-GFP vector with the CaMV 35S promoter driving GFP alone was used as a negative control. A transient expression assay was performed in leaves of Nicotiana benthamiana. PavMYC2-GFP fusion construct revealed GFP fluorescence in nucleus, compared with empty PHB-GFP vector. To better understand genetic relationship between PavMYC2 and other reported MYC2-like genes, we performed the phylogenetic tree analysis, which indicated that PavMYC2 was closely to PpsMYC2 and PyMYC2 from P. pseudocerasus and P. yedoensis, suggesting a conserved function with these evolutionarily closer MYC2 homologs. To investigate the possible roles of PavMYC2 in the development of sweet cherry, we performed the relative expression levels from various tissues of sweet cherry by using qRT-PCR analysis. PavMYC2 expression in sweet cherries was observed in vegetative and floral tissues, but much higher level in flower buds with 4.8-, 4.9-, 8.8-, 37.4-fold than that in young leaves, stems, roots and flowers. As a result, the highest transcriptional level of PavMYC2 occurred in floral buds, indicating that PavMYC2 might play an important role in the development of floral buds.The seasonal expression level of PavMYC2 was higher during the stage of summer and autumn growth in floral buds, then gradually decreased, maintained a certain level of expression in winter, and reached the lowest level in spring. The cis-acting element analysis showed that the PavMYC2 gene promoter contained a large number of light-responsive elements, hormone-responsive elements and temperature stress-related elements. We performed BiFC assays to detect protein-protein interactions. The BiFC assays indicated that PavMYC2 interacted with PavJAZ1/2/3 in leaves of Nicotiana benthamiana, which had similar expression patterns with PavMYC2, suggesting that PavMYC2 and PavJAZs may co-regulate the temperature stress and flowering.【Conclusion】A PavMYC2 gene was cloned and highly expressed in summer and autumn stages, and thereafter declined gradually, maintained a certain level of expression in winter, and reached the lowest in spring. This study lays the foundation for further investigation on the role of MYC2 in sweet cherry buds in response to temperature stress and regulation of flowering process.