| The F-box protein family is a large family that is characterized by conserved F-box domains of approximately 40 to 50 amino acids in the N-terminus. F-box proteins participate in diverse cellular processes, such as development of floral organs, signal transduction and response to stress, primarily as a component of ubiquitin pathway system. Identification and analysis F-box gene family in genome-wide is of vital importance for discovering novel genes and studying gene functions. Apple(Malus domestica) is an important economic crop. Studies on apple F-box gene family are still limited. In this study, using a global search of the apple genome, 517 F-box protein-encoding genes(F-box genes for short) were identified. These genes were subdivided into different groups according to their functional domains. Chromosomal localization, expression profile, stress response et al. were further analysed. The results can provide useful information for elucidating the function of F-box genes in apple. The main results are as follow:(1) Two strategies and two step screening were used to obtain 517 F-box genes in apple genome.In the first strategy, all of the predicted sequences of F-box proteins from Arabidopsis, maize and rice were used as queries to search the apple peptide database using BLASTp. To obtain credible results, the E-value cut-off was set to 0.001. For the second strategy, based on the characteristics of the amino acid sequences, a Hidden Markov Model(HMM) profile of the F-box domain was established by the pfam website. This HMM profile of the F-box domain was used as a query to search in the apple peptide database. Using this strategy, 571 proteins that potentially contained the F-box domain were obtained. To further verify the F-box domains that were presented in these proteins, all of the sequences that were identified by the above two strategies were submitted to the Interpro Database to perform checking. Finally, 517 F-box proteins and their relative encoding genes were obtained.(2) F-box proteins identified were subdivided into 12 groups according to the functional domains.F-box proteins often contain additional functional domains. According to the characterization of known functional domains, F-box genes were subdivided into 12 groups, which suggests the different potential functions or processes that they were involved in. Among these domains, the galactose oxidase domain was analyzed for the first time in plants, and this domain was present with or without the Kelch domain.(3) Apple F-box genes contain several conserved amino acids.The sequences of the F-box domain from the above 517 proteins were submitted to the MEME website. A conserved F-box motif of 40 amino acids in the apple genome was identified, in which the Leu, Pro, Val and Trp residues at positions 4, 5, 27, and 31 were highly conserved. The Leu(Phe), Leu(Val), Pro, Cys(Ser), Lys(Arg), and Phe(Leu) residues at positions 13, 16, 17, 28, 29, and 40, respectively, were conserved..(4) Analysis on chromosomal distributions of apple F-box genes.Among the 517 F-box genes, 492 genes can be mapped to all the 17 apple chromosomes. Results analysis indicated that tandem gene duplication occurred in apple F-box genes. Large numbers of apple F-box genes were located in homologous regions, which was thought to be formed by a genome-wide duplication event. The tandem duplication of genes and genome duplication was two important ways for F-box genes to extend in the apple genome.(5) Spatial expression profile analysis revealed that F-box genes have organ-specific expression.Spatial expression profile was analyzed using transcriptome data downloaded from GEO database. Expression information of 517 F-box genes were extracted and clustered using cluster3.0 software. These genes were divided into 7 groups according to their different expression level in different organs. Ten genes were selected from each group to examine their expressions in different organs using gene-specific primers. The expression tendency of most F-box genes in different organs was consistent with the data in the transcriptome profile.(6) Analysis on spatial expression profile of F-box genes in group FBG and FBKG.Group FBG and FBKG contained genes encoding proteins with galactose oxidation domain. According to their different expression level in different organs, these genes were divided into 2 groups. F-box genes in the two groups were mainly expressed in seeds, seedling, flower and fruit, indicating the potential roles in these organs.(7) Apple F-box genes were involved in fruit ripening and abiotic stress response.From a fruit ripening expression profile, 166 F-box genes were identified. The expressions of most of these genes changed little during maturation, but five of them increased significantly. Using qRT-PCR to examine the expression of F-box genes encoding proteins with domains related to stress, the results revealed that F-box proteins were up- or down-regulated, which suggests that F-box genes were involved in abiotic stress. |
