Identification and expression profile analysis of oleosin gene family in Chinses hickory and pecan

Abstract: 【Objective】Lipid droplet (LD) plays an essential role in seed germination. Oil body proteins (oleosin) are a kind of proteins localized on the surface of LD, which participates in the regulation of LD biogenesis, morphology homeostasis, and stabilization. Chinese hickory (Carya cathayensis) is an arbor with high oil content in its embryo. However, the molecular mechanism of high oil accumulation in hickory embryos remains unclear. This study aimed to systematically investigate the oleosin genes in the Chinese hickory and analyze their expression patterns.【Methods】Based on the Pfam database, the oleosin genes in Chinese hickory (C. cathayensis) and pecan (C. illinoinensis) were identified and characterized in this study. Then, gene structures and conserved motifs were analyzed by TBtools, and the cis-acting elements of oleosin gene promoter regions were performed using the Plant CARE database, and phylogenetic analysis of oleosin gene family from C. cathayensis, C. illinoinensis, Arabidopsis thaliana, Glycine max, Arachis hypogaea and Brassica napuswere conducted using MEGA 7.0 software (Bootstrap=1000). Moreover, the expression patterns during different developmental stages of the em-bryo were analyzed. Furthermore, subcellular localization of oleosin proteins was conducted.【Results】 The results showed that seven and eight oleosin genes containing a conserved domain“proline knot” were identified in Chinese hickory and pecan, respectively. The oleosin protein molecular weights ranged from 14.54 to 17.51 ku, and the isoelectric point of oleosin protein ranged from 9.33 to 10.36. The gene structure analysis showed that all members of the oleosin genes in hickory and pecan contained one exon but not intron. Four conserved motifs were found in the oleosin gene family. Based on the analysis of the conserved motifs, CCA0791S0002, CIL0938S0150 and CIL0252S0009 might share a similar function. The results of multiple amino acid sequence alignment showed that oleosin proteins of hickory and pecan contained a typical and highly conserved“proline knot”-PX5SPX3P-. The“proline knot”domain is a special structure in the oil body, and it is responsible for oil body localization. Lots of cis-acting elements were found in the upstream 2000 bp of the oleosin genes, including gibberellin (GA), abscisic acid (ABA), MeJA, and a variety of environmental stresses, indicating that the signals of abiotic stress and hormones might regulate oleosin genes expression. According to the phylogenetic analysis result, all oleosins genes were clustered into the U-Oleosin, SL-Oleosin, and SH-Oleosin subgroups. The SL-Oleosin is the largest subgroup, accounting for about 1/2 of the oleosin gene members of hickory and pecan. In the SL-Oleosin subgroup, the oleosin genes of hickory and pecan were distributed in two branches. CCA0791S0002, CIL0938S0150 and CIL0252S0009 were classified into one branch, and CCA0520S0025, CIL1494S0045, CCA0779S0183 and CIL0922S0190 were classified into another branch. In the U-Oleosin subgroup, the oleosin genes of hickory and pecan were also distributed in two branches: CCA0674S0096 and CIL0212S0015 clusters were distributed in the same branch, while only CIL1212S0020 distributed in another branch. In the SH-Oleosin branch, three oleosin genes in hickory and two oleosin genes in pecan were clustered to one branch. Moreover, the expression patterns of the oleosin genes at different stages of embryo development in hickory (S1, S2, S3, S4 and S5) were analyzed using the transcriptome database. The results showed that the oleosin genes were highly expressed during the different developmental stages of the embryo. CCA0674S0096, CCA0779S0183, CCA0514S0208 and CCA0520S0025 had the same expression trend, which could be classified into one group. The expression trends of CCA0791S0002, CCA0656S0072 and CCA0656S0074 could be clustered into one category. Compared with the S1 stage, all oleosin genes were up-regulated at different stages of embryo development. The mRNA expression levels of CCA0656S0072 and CCA0656S0074 were significantly up-regulated from S4 to S5 stage. The mRNA expression level of CCA0520S0025 and CCA0791S0002 significantly increased from S2 to S3 stage. It was noted that the oil content continuously raised during the developing process of the embryo and showed a sharp increase from S2 to S3. The qRT-PCR analysis results showed that CCA0520S0025 and CCA0791S0002 were responded to ABA and low-temperature stress. Most oleosin genes showed an up- regulated expression trend under the ABA treatment. Three genes (CCA0791S0002, CCA050S0025 and CCA0779S0183) reached peak expression 2 h after the ABA treatment, and CCA0656S0074 showed a continuous up- regulation after the ABA treatment, and two genes (CCA0514S0208 and CCA0674S0096) showed a down-regulation 6 h after the ABA treatment, and then reached the peak 12 h after the treatment. Under the low-temperature stress, CCA0791S0002, CCA0520S0025 and CCA0514S0208 showed an up- regulated expression 2 h, 4 h and 6 h after the treatment, respectively. Finally, the pBI221- UBQ10- CCA0520S0025- GFP and PBI221- UBQ10- CCA0791S0002- GFP expression vectors were completed. The subcellular localizations were performed through transient expression using protoplasts isolated from the Arabidopsis suspension culturecells. And the results showed that CCA0520S0025 and CCA0791S0002 were colocalized with AtOLEO2-RFP, an Arabidopsis oil body gene.【Conclusion】The oleosin gene family of Chinese hickory and pecan had similar gene structure and conserved motifs. In addition, CCA0520S0025 and CCA0791S0002 might play an important role in the oil accumulation in hickory embryo.