Abstract:

【Objective】The transcriptomics and bioinformatics methods were used to carry out the genome-wide potential interacting MdARFs and MdIAAs pairs screening, so as to build a foundation for clarifying the function of related genes and elucidating the molecular mechanism underlying auxin-regulated apple fruit size.【Method】 Two apple genotypes, Royal Gala (WT) and 35S::miRNA172p transgenic Royal Gala (miR172OX ) were used as test materials in this study. Fruit materials from different developmental stages and tissue types were collected for both genotypes and were subjected to transcriptome sequencing. Clean reads were aligned to the reference genome and the differential expression analysis was performed. Based on the transcriptome data obtained by sequencing the transgenic small fruit and wild-type large fruit, the pairwise expression analysis was performed across MdARFs and MdIAAs families. The amino acid sequences of 23 ARFs and 34 Aux/IAAs were downloaded from Arabidopsis genome, and 21 ARFs and 25 Aux/IAAs were downloaded from tomato genome, which were further compared with candidate MdARFs and MdIAAs to construction phylogenetic trees. The MEME and TBtools were used to carry out the Motif analysis for candidate MdARFs and MdIAAs. Pairs with high interacting possibilities were further confirmed by a protein-protein interacting network constructed in apple to finalize combinations with the highest probability of involvement in fruit development. 【Result】 The whole fruit at 2 weeks post full bloom and the fruit skin, fruit flesh and fruit core at 4 WPFB were collected from WT and miR172OX, respectively. To achieve research objectives, transcriptome sequencing was carried out. A total of 178.19 Gb paired-end reads of 125 bp/150 bp were generated. All indexes indicated that the three biological replicates had highly consistent transcriptome profiles across all tissue types. FPKM values in at least one library was over 2 were used as a standard to eliminate the low expressed genes, so a total of 38MdARFs and 27 MdIAAs were expressed. In our fruit developmental transcriptome data, eight pairs of MdARF-MdIAA were obtained through Pearson correlation analysis, whose Pearson correlation coefficient was over 0.9 or below -0.9. The systematic phylogenetic analysis showed that MdARF6 and MdARF19 belonged to the same branch with AtARFs, which played a role in transcription activation, while MdARF2, MdARF4, and MdARF9 were closely related to transcriptional inhibitory AtARFs. Motif analysis results showed that both the candidate MdARFs and MdIAAs proteins contained Motif 2 and Motif 5, which were corresponded to the conserved domains Motif IV and Motif III in the IAA protein, respectively. After homolog mapping inspection with Arabidopsis, two potential MdARF-MdIAA interacting pairs were selected for future functional identification. 【Conclusion】Among apple MdARF and MdIAA family members, eight pairs of MdARF-MdIAA showed significant correlations in terms of their expression patterns during fruit development. Further homology mapping confirmed two pairs of them, including MdARF4-MdIAA17 and MdARF4-MdIAA19, were most likely to participate in the regulation of apple fruit development through mediating auxin signal transduction.

Key words: apple, ARF, Aux/IAA, interacting-pair screening