Identification and expression analysis of the CM family gene in walnut (Juglans regia L.)

Abstract:【Objective】It is estimated that about 20% of the carbon fixed by green plants goes through the shikimic acid pathway (SAP), and provides the carbon skeleton for secondary metabolites such as aromatic amino acids, vitamins, lignins, phenols and aromatic substances. Hence, SAP is regarded as the bridge connecting sugar and secondary metabolism in both plants and microorganisms. Chorismate mutase (CM) is a constitutional enzyme of the SAP pathway, catalyzing the conversion of chorismate to prephenate. In the present study, genes of the CM family were first identified from the genomic data of walnut (Juglans regia L.), aiming to uncover their physicochemical properties and genetic features. 【Methods】Protein identification was performed by searching the genomic data using the Hidden Markov Model (HMM) model of the CM family protein. The Arabidopsis thaliana AtCM1 protein was also used as the seed to BLAST against the walnut proteins, in order to further confirm the searching result. The walnut genome data was downloaded from the Ensembl Plants database. The non- redundant se-quences were re- confirmed by querying the SMART and CDD databases and used as sequences for downstream analyses. The physicochemical parameters, including gene length, molecular weight, isoelectric point, amino acid number, instability coefficient, signal peptide, chromosome location, transmembrane structure, hydrophobicity and subcellular location, were obtained by calculation and prediction using bioinformatic tools. For display of gene structure, structure information was extracted from the sequence annotation file, and displayed by using TBtools. We also predicted the cis-acting elements existing in the promoter regions by querying the PlantCARE database, and the location of the motifs was displayed using GSDS. The phylogenetic analysis of plant CM proteins was performed using the neighbor-joining (NJ) method with 1000 bootstrap replicates (implemented in the MEGA11 software). For quantification of gene expression by qPCR, the primers of CM genes were designed using the Oligo Architect Online tool, and the walnut 18S gene was selected as the internal reference gene. Real time fluorescent quantitative PCR was then conducted to quantify and analyze the gene expression in different tissues (leaf, stem, fruit stalk, green husk).【Results】After HMM and BLAST search, seven CM proteins were identified from the walnut genome. All the CMs contained the CM-2 domain, which was specific to the family. Therefore, these seven genes were identified as members of the CM family. Analyses of physicochemical properties showed that all 7 CMs were hydrophilic proteins except for JrCM5. No signal peptide was detected in walnut CMs, except for JrCM5. Most of the CMs identified in other species had no signal peptide, such as Petunia hybrida PhCM1 and Salvia miltiorrhiza SmCM1. It was speculated that walnut CMs had similar property and function like other homologues. The prediction of protein subcellular localization showed that walnut CMs were all located in the chloroplast, which was consistent with that of PhCM1. Therefore, the functioning place of the CM homologues could probably be in the chloroplast. The CMs might play active roles in plant defense by affecting the synthesis of secondary metabolites, such as salicylic acid and flavonoids. The gene location analysis showed that these 7 CM genes were distributed on 6 chromosomes. Each gene contained 5-6 exons, and their structure was conservative. All the 7 CMs contained no transmembrane structures, except for JrCM4. The results of protein three- dimensional structure prediction showed that there was a high similarity between the members of the walnut CM family proteins and the template AtCM1 protein. A diverse set of cis-acting motifs were detected in the promoter region of CM genes. The CAAT box and TATA box were constantly found in all the promoters. In addition, a total of 296 elements, which could be grouped into seven major categories were detected. These cis- elements could be grouped into 7 categories, including the light response, hormone response, stress response, transcription factor binding site, growth and development response, and circadian rhythm regulation. The phylogenetic analysis showed that the plant CM proteins could be divided into four groups, and the CMs were clustered in branch Ⅱ and Ⅳ. Wherein, JrCM2 and JrCM5 were closely related to Populus tomentosa CM Potri.T174200 protein; JrCM6, JrCM7 and JrCM3 were closely related to grape CM GSVIVGO1010091001 protein; JrCM1 and JrCM4 had close relationship with Phalaenopsis Aphrodite CM PEQU04269. The results of qRT-PCR analysis showed that 7 genes were expressed in walnut leaves, stems, fruit stalks and green husks, and their expression was tissue specific.【Conclusion】In this study, seven CM family genes were identified from the walnut genome. The proteins of walnut CM family had physicochemical properties and three-dimensional structures similar to those of their homologues. A variety of cis-acting elements were detected in the promoter region of these 7 genes, implying responsiveness to multiple signals. The expression of CM family genes was universally detected in different tissues. The gene expression profiling of CM genes is not only helpful for better understanding of the generation of secondary metabolites (variousamino acids, phenols and antioxidant active substances), but also benefitial for the regulation mechanism study of CM proteins. The biosynthetic pathway of plant polyphenols is very complex, involving a wide range of enzymes, and chorismate mutase is only one of them. At present, the functional mechanism of this family is still not fully elucidated, and we will continue to carry out downstream experiments to uncover the molecular mechanism of the shikimic acid pathway regulation.