Identification and analysis of AP2 subfamily transcription factors in sweet orange (Citrus sinensis)

Abstract:【Objective】AP2 subfamily transcription factors have the function of regulating the development of plant seeds, leaves, flowers, roots, stems and other organs and the responding to abiotic and biotic stresses. In this study, we performed gene identification, bioinformatics analysis, and gene expression analysis of the AP2 subfamily in order to provide a theoretical basis for studying the biological functions of AP2 subfamily genes in sweet orange (Citrus sinensis).【Methods】The bioinformatics analysis was used to screen and identify AP2 subfamily genes in sweet orange. The gene expression patterns in various tissue and abiotic stresses were analyzed by qRT- PCR.【Results】14 AP2 subfamily genes were identified from the sweet orange genome database using bioinformatics methods. According to the analysis of protein physicochemical properties, the AP2 subfamily proteins of sweet orange were all unstable hydrophilic proteins. These proteins were primarily localized in the nucleus, which was consistent with their function and properties as transcription factors. The results of phylogenetic tree analysis showed that AP2 subfamily proteins of sweet orange could be divided into three evolutionary branches, including the ancient clade (CsRAP2-5, CsRAP2-6, CsRAP2-9, CsRAP2-2 and CsRAP2-13), the intermediate clade (CsRAP2- 1, CsRAP2- 7, CsRAP2- 11 and CsRAP2- 14) and the modern clade (CsRAP2-2-CsRAP2-4, CsRAP2-8 and CsRAP2-10). In the phylogenetic tree of plant AP2 subfamily proteins, there were significant interspecific differences in the number of AtAP2, OsAP2, CcAP2 and CsAP2 protein members clustered in the same group. The AP2 subfamily proteins of sweet orange were more closely related to the homologous proteins of Clementine orange than the homologous proteins ofArabidopsis and rice. The protein conserved motif analysis showed that the AP2 subfamily proteins contained conserved elements of Motif 3, Motif 4, Motif 2, Motif 5 and Motif 1. In addition, each group in the orange AP2 subfamily protein phylogenetic tree had its specific conserved elements. These results indicated that the members of the same subgroup of AP2 subfamily proteins in sweet orange were highly conserved, which also would reflect the reliability of the phylogenetic analysis results. The number of introns and exons of AP2 subfamily genes in sweet orange was 5-9 and 6-10. The promoter sequences of AP2 subfamily genes in sweet orange contained cis-acting elements such as endogenous hormone response, growth and development and abiotic stress response, indicating that the subfamily genes might have the function of regulating plant growth and development in response to plant hormones, light and abiotic stresses. The results of collinearity comparative analysis showed that sweet orange had more AP2 subfamily homologous gene pairs with Arabidopsis and apple compared with rice and maize. These results indicated that compared with monocots, the AP2 subfamily genes of sweet orange had more homologous genes and closer relatives with dicots. The prediction of protein secondary structure showed that the AP2 subfamily proteins of sweet orange had the highest proportion of α-helix and random coil. The tssue- specific expression analysis showed that AP2 subfamily genes were expressed in three tissues: leaves, stems and roots, with higher expression levels of the CsRAP2-3 and CsRAP2-6 in the roots and higher expression levels of the CsRAP2-7 in the stems. In order to further verify the response of AP2 subfamily genes to abiotic stresses in sweet orange, the qRT-PCR was used to detect the expression pattern of AP2 subfamily genes in the leaves of sweet orange under simulated drought (20% PEG6000), high salt (250 mmol · L- 1 NaCl), abscisic acid (100 μmol · L- 1 ABA) and low temperature (4 ℃) stresses. The results showed that the expressions of the CsRAP2-1, CsRAP2-2, CsRAP2-8 and CsRAP2-10 were generally down-regulated under simulated drought, high salinity, ABA and low temperature treatments. The expression of the CsRAP2-12 was down- regulated after high-salt, ABA and lowtemperature treatments, and down-regulated at 1-12 hours after simulated drought treatment, but the expression level was higher at 24 h than that before treatment. Except for these five genes, the expression levels of the other 9 AP2 subfamily genes in sweet orange showed an upward trend after drought treatment. The CsRAP2-3, CsRAP2-4, CsRAP2-6, CsRAP2-7, CsRAP2-9, CsRAP2-11 and CsRAP2-13 were induced by high salt stress. After ABA treatment, only the expression of the CsRAP2-7 showed an upward trend. Except for the CsRAP2-13, the expression levels of the other 13 genes showed a down-regulated trend after low temperature treatment. These results indicated that the expression of most AP2 subfamily genes in sweet orange increased under drought and high salt stress, and decreased under ABA and low temperature treatments.【Conclusion】The AP2 subfamily genes of sweet orange were identified and analyzed in detail at the genome-wide level. The tissue expression characteristics of this subfamily genes and their responses to abiotic stress were studied, which would provide a basis for the subsequent study of the function of these genes in regulating the citrus responses to abiotic stresses.