Gene cloning and protein characterization analysis of EjWRKY15 in loquat

Abstract:【Objective】WRKY transcription factor is a plant-specific super-gene family, and the WRKY protein plays an important role in signaling pathways that regulate plant growth and development, biot- ic and abiotic stress defense, hormone transduction and other biological metabolic processes. In previ- ous study, 33 sequences with a typical WRKY domain have been isolated and identified. It was found from the research basis of fruit sunburn transcriptome that the transcription factor EjWRKY15 of theWRKY gene family was highly up-regulated under sunburn stresss, indicating that the EjWRKY15 gene was responsible for the resistance to the sunburn reaction and the synthesis of functional genes played an important role in loquat [Eriobotrya japonica (Thunb.) Lindl.]. The main objective of this study was to clone the gene EjWRKY15 and reveal its tissue expression patterns and protein characteristics in lo- quat【. Methods】The full-length ORF sequence of the EjWRKY15 gene was obtained by sequencing, and the amino acid sequence of the EjWRKY15 gene was compared based on NCBI database, then the genes with high homology were screened and downloaded. MEGE X software was used to construct phyloge-netic tree. The molecular weight, isoelectric point, liposoluble index, instability index, average hydro- philicity and other physical and chemical properties of encoding protein were analyzed by Protparam Protparam software. The potential phosphorylation sites of the EjWRKY15 protein in loquat were pre- dicted by NetPhos v3.1 online tool. SOPMA and SWISS-MODEL were respectively used to predict sec- ondary structure and tertiary structure online; the transmembrane regions and signal petide were also an- alyzed using TMHMM Server v.2.0 and SignalP 4.1 Server. Plant-mPLoc v1.0 was used to predict sub- cellular localization and then pBWA(V)HS-EjWRKY15-GLosgfp vector was constructed to verify the subcellular localization【. Results】The cloning and sequencing results showed that the EjWRKY15 gene contained 951 bp of open reading frame, which encoded 316 amino acids and had typical structural characteristics of WRKY family. Phylogenetic tree analysis results showed that the EjWRKY15 sequence had 95% homology with apple WRKY61 sequence, indicating the EjWRKY15 might have the closest re- lationship with Malus domestica from an evolutionary perspective.The results of real-time fluorescence quantitative PCR showed that the EjWRKY15 gene had tissue expression specificity, with higher abun- dance expression in the roots than in the fruits, suggesting that this gene was mainly expressed in the roots. The results of physical and chemical properties of the EjWRKY15 protein showed that the molecu- lar weight, isoelectric point, fat solubility index, instability index and average hydrophilicity were 35.79, 5.09, 56.45, 69.72 and-0.598, respectively, indicating that it was a hydrophilic protein. It was found that the EjWRKY15 proteins had 33 phosphorylation sites in total, containing serine (Ser), threo- nine (Thr) and tyrosine (Try), the number of them was 25, 6, 2, respectively. The results of protein sec- ondary structure prediction showed that the EjWRKY15 protein was mainly a-helix and random curl, with the proportion of 30.70% and 56.65% respectively, followed by 8.27% of extension chain and 4.43% of β corner. At the same time, the tertiary structure analysis showed that the domain of this pro- tein was mainly composed of four β-folds, β-fold 1 was WRKYGQK, β-fold 2 was RSYYKC, β-fold 3 was GVRKHVER and β- fold 4 was AVVTTY. In addition, prediction results confirmed that theEjWRKY15 protein did not contain signal peptide and was not a secretory protein, which did not contain transmembrane region at the same time. The results of subcellular localization prediction showed that the EjWRKY15 gene was located in the nucleus, the above result also verified the accuracy by tobacco instantaneous expression system, which was consistent with this gene as a transcription factor Control downstream gene expression【. Conclusion】The results of cloning and protein characterization of theEjWRKY15 gene in loquat will laid a good foundation for further study on the molecular biological func- tion of the EjWRKY15 gene in different growth and development stages of loquat and the mechanism of response to adversity regulation.