Identification and functional analysis of PEPC gene family in Hevea brasiliensis

【Objective】 This study aimed to identify members of the phosphoenolpyruvate carboxylase （PEPC） gene family of whole genome in Hevea brasiliensis and analyze their functions， thereby providing reference for studying the regulation mechanism of PEPC gene family functions in rubber biosynthesis. 【Method】 PEPC gene family members were identified from Hevea brasiliensis genome using Arabidopsis thaliana PEPC protein sequences as probes， and their physicochemical properties，conserved domains， and subcellular localization of the encoded proteins were analyzed. Transcriptome data were used to perform PEPC gene expression analysis. Protein interactions between HbPEPC3 and HbSUS3 were validated using yeast two-hybrid and bimolecular fluorescence complementation assays. 【Result】 Six PEPC genes were identified from Hevea brasiliensis genome， which were named HbPEPC1-HbPEPC6， encoding 692-1066 amino acids， with the theoretical isoelectric point of 5.56-6.16，and the instability coefficients were all above 40.00， indicated that they were unstable proteins， and the grand average of hydrophilicity/hydrophobicity coefficients ranged from -0.441 to -0.351， all belonging to hydrophilic proteins. The six PEPC family members from Hevea brasiliensis were divided into two subfamilies. HbPEPC1 and HbPEPC5 were clustered in the BPTC clade， belonging to bacterial-type PEPCs， while the other four members were clustered in the PTPC clade， belonging to plant-type PEPCs. In these two clades， the PEPCs of Hevea brasiliensis and cassava were clustered together， which was consistent with the taxonomic status of Hevea brasiliensis and cassava， as both belonged to Crotonoideae of Euphorbiaceae. Analysis of gene structure revealed that the gene structure of PEPCs was consistent with their phylogenetic classification， with the BTPC clade containing more introns. The six PEPC gene family members were distributed on six Hevea brasiliensis chromosomes； collinearity was found between HbPEPC2 and HbPEPC3， between HbPEPC4 and HbPEPC6 in the PTPC clade，while collinearity was found between HbPEPC1 and HbPEPC5 in the BTPC clade. The HbPEPC3 exhibited a relatively high expression in the bark， with a down-regulated expression trend during the tapping process. The HbPEPC5 gene in the BTPC clade showed high expression in leaves and down-regulated trend during the tapping process， and the HbPEPC5 gene showed down-regulated trend under ethylene treatment. The HbPEPC3 had interaction with HbSUS3， and it was collinear with the AtPEPC4 gene in Arabidopsis thaliana， suggesting that it had similar functions. 【Conclusion】 Six PEPC gene family members in Hevea brasiliensis distribute in the two subfamilies， BTPC and PTPC. The PTPC subfamily member HbPEPC3 shares high local collinearity with PtPEPC1 and AtPEPC4 genes in model plants， and shows expression of high abundance in the bark. Protein interactions are found between HbPEPC3 and HbSus3， suggesting that HbPEPC3 plays an important role in rubber biosynthesis， particularly in carbon source supplementation.