Functional analysis of the COP9 subunit FaCSN5 during strawberry fruit development

Abstract：【Objective】The study aimed to investigate the function of strawberry (Fragaria × ananassa) CSN5 (constitutive photomorphogenic signalosome subunit 5) during strawberry fruit development. 【Methods】Benihoppe strawberry was used as experimental material. Firstly, based on the transcriptome data of the five developmental stages (SG, LG, Wt, IR and PR) of strawberry fruit, a gene with increased expression level during fruit development from the large green fruit was screened. Because it contained MPN conserved domain and had 100% sequence similarity with diploid strawberry FvCSN5 (XM_004291211), the gene was named FaCSN5. Total RNA was extracted from the samples using a total RNA extraction kit (Guangzhou Magen Biotechnology Co., Ltd.), and the cDNA was synthesized by reverse transcription using Hi fair ® Ⅲ 1st Strand cDNA Synthesis Super Mix for qPCR (ShanghaiYeasen Biotechnology Co., Ltd.) kit, and then the full-length CDS sequence of FaCSN5 was obtained by PCR. Secondly, some bioinformatics techniques were used in this study. The molecular formula, molecular weight, isoelectric point, and fat solubility index of encoding protein were analyzed in the Prot Param website. The conserved domain of FaCSN5 was analyzed by the NCBI website and Blast tool. The transmembrane domain of FaCSN5 protein was analyzed by TMHMM 2.0. The secondary and tertiary structures of FaCSN5 were analyzed online using NPS and Swiss Model. The phylogenetic tree of FaCSN5 homologous genes was constructed by MEGA11 software. Thirdly, the spatiotemporal expression levels of FaCSN5 were detected by RT-qPCR using Agrobacterium-mediated transient transformation of strawberry fruits, and the phenotypes were observed and used to detect the expression levels of the FaCSN5 gene. The cis-acting elements of FaCSN5 gene promoter were analyzed by the online software Plant CARE. The induction of FaCSN5 gene expression by exogenous hormones was detected by disc incubation and exogenous hormone treatment experiments. The subcellular localization was observed by Agrobacterium-mediated transient transformation of tobacco mesophyll cells. Finally, the fulllength CDS sequence of FaCSN5 was constructed into pET30a vector by homologous recombination, and the pET30a-FaCSN5 fusion expression vector was obtained induced and purified. Western Blot was used to detect FaCSN5-His target protein by SDS-PAGE and Anti-His antibody.【Results】Evolutionary tree analysis showed that FaCSN5 was highly homologous to FvCSN5 and rosa CSN5b, with similarity rates of 100% and 94.24% , respectively. The base and amino acid sequence similarity between the cloned FaCSN5 and the eight gene sequences of Yanli strawberry reached 98.97% and 99.35%, respectively. Bioinformatics analysis showed that the coding sequence of FaCSN5 was 1080 bp, encoding 359 amino acids. The analysis of physicochemical properties of amino acids showed that the molecular formula of FaCSN5 was C1797H2773N471O558S14, the molecular weight of the protein was 40.35 ku, and the isoelectric point (pI) was 4.93, which was an acidic protein. The protein contained 48 negatively charged amino acid residues (Asp + Glu) and 31 positively charged amino acid residues (Arg + Lys). The instability coefficient was 41.41, and the average hydrophilicity is -0.421. It was inferred that the protein should be an unstable hydrophobic protein. Conserved domain analysis showed that FaCSN5 had a conserved MPN domain. Phylogenetic tree analysis showed that FaCSN5 had high homology with FvCSN5 and rose CSN5b, and the similarity rates were 100% and 94.24%, respectively. The pET30a-FaCSN5 fusion expression vector was constructed for prokaryotic expression in Escherichia coli. The results of SDS-PAGE and Western Blot showed that the size of FaCSN5-His target protein was about 66 ku. The transient expression of Nicotiana benthamiana showed that the FaCSN5-GFP fusion protein was localized in the nucleus and cytoplasm. RT-qPCR analysis showed that the expression level of FaCSN5 was the highest in the root, followed by FR, PR, stem, leaf, and flower. The expression level of FaCSN5 was the lowest in the seed, and the expression level in the root was 8 times higher than that in the seed, indicating that FaCSN5 had tissue specificity. During fruit development, the expression level was the highest at the Full red stage and the lowest at the De-greening stage. From the Degreening green stage, the expression level increased with fruit development, indicating that FaCSN5 might be involved in the development of strawberry fruit. Agrobacterium- mediated transient infestation of strawberry fruit with FaCSN5 overexpression could promote strawberry fruit ripening; silencing FaCSN5 expression inhibited strawberry fruit ripening. The FaCSN5 promoter contained cis-acting elements in response to methyl jasmonate and gibberellin. After treatment with MeJA and GA, the expression level of FaCSN5 gene was slightly lower than that of the control group after 1 h of MeJA treatment. After 2-5 h of treatment, the expression level of FaCSN5 gene was higher than that of the control group. The expression level ofFaCSN5 gene was higher than that of the control group at 1-5 h after GA treatment, and the expression level was the highest at 1 h after treatment. After ABA treatment, the expression level of FaCSN5 gene was higher than that of the control group, and the expression level was the highest after 3 h of treatment. The results showed that the expression level of FaCSN5 gene was induced by MeJA, GA, and ABA, to various degrees.【Conclusion】The protein height of FaCSN5 was about 66 ku, which was localized in the nucleus and cytoplasm. The expression of FaCSN5 was induced by abscisic acid, methyl jasmonate, and gibberellin. FaCSN5 might promote strawberry fruit ripening through multiple hormonal regulations.