Identification and bioinformatics analysis of CHS genes in different phenotypic leaves of natural hybrid progenies of red-kernel walnut

Abstract: 【Objective】Anthocyanin is a widely distributed flavonoid metabolite, which plays important roles in setting color in plant tissues and organs. Chalcone, the basic skeleton of flavonoids, is the key position in the whole flavonoids synthesis pathway, and its synthetase (CHS) is the first key enzyme in anthocyanin synthesis pathway, which determines the anthocyanin synthesis direction and the final prod-uct numbers. In this research, the function of JrCHS genes in the anthocyanin biosynthesis was studied to provide a theoretical basis in the molecular mechanism of anthocyanin synthesis in red-kernel walnut.【Methods】By comparing the results of transcriptome sequencing with walnut genome database,  four CHS genes, JrCHS1- JrCHS4, and corresponding Chalcone synthetase as functional annotation were selected from different phenotypes (red leaf and green leaf) of natural hybrid progenies of red-ker-nel walnut in three developmental stages. The corresponding sequences, physicochemical properties and structural functions were analyzed by bioinformatics. The basic information of CHS genes was obtained from NCBI database. The isoelectric points and molecular weights of CHS proteins were predicted by ExPASy tool, and the subcellular localization of CHS proteins was analyzed by Cell-PLoc online soft-ware. Tbtools software was used to identify the structure of CHS genes and chromosomal position. MEME online software was used to analyze the conserved motifs of CHS proteins. ProtScale and TM-pred software were used to analyze hydrophobicity and protein transmembrane, and SOPMA and SWISS-MODEL were used to predict protein secondary and tertiary structures. The sequence of multi species CHS proteins was analyzed by MEGA 7.0 software, and NJ method was used to construct phylo-genetic tree. The upstream 2000 bp fragment of CHS genes was selected to analyze the cis-acting ele-ments of promoter by the PlantCARE software. The RNA-seq data of different phenotypes of natural hy-brid progenies of red-kernel walnut were analyzed, and the CHS gene expression map was constructed by TBtools. Finally, the expression levels of all genes were analyzed by qRT-PCR.【Results】There were significant differences in color phenotype and anthocyanin contents between the two types of leaves at different development stages. Four JrCHSs were located at Chr 1, 2, 3 and 7, respectively, and the physi- cal and chemical properties of the corresponding proteins were similar, which were mainly distributed in the cytoplasm. Four genes only contained one intron structure, and the difference in distribution was small. The number and location of protein conserved motifs were the same, all of which belonged to hy- drophilic acid protein, without transmembrane structure. The proportion of α-helix in all protein second- ary structure was the highest, and the three-level spatial structure models were composed of single helix protein subunits. Multi species phylogenetic analysis showed that four JrCHS proteins had high homolo- gy with known anthocyanin synthesis related proteins such as CnCHS, RsCHS, VaCHS, PtCHS, LcCHS, DlCHS, MeCHS, PgCHS1 and PgCHS2, and it was speculated that their functions were also similar. Using PlantCARE, hormone response elements, stress response elements, light response ele-ments and transcription factor binding site elements were found in JrCHS promoters, and all the promot-er sequences contained multiple MYB and MYC transcription factor binding sites. Four JrCHS genes were expressed at different stages of leaf development with different phenotypes, and in the first stage, the expression levels in the red leaf were significantly higher than those in the green leaf, which was pos-itively correlated with the color phenotype and anthocyanin contents, and there was also different expres-sion in other stages. qRT-PCR analysis further confirmed the above results and verified the reliability of transcriptome data.【Conclusion】The structure and function of the four JrCHS genes and their encoded proteins were similar, which may be due to the differentially expressed genes related to anthocyanin me-tabolism in different phenotype leaves of natural hybrid progenies of red-kernel walnut.