Genome-wide characterization and expression analyses of the auxin/indole-3-acetic acid(Aux/IAA)gene family in Longan (Dimocarpus longan Lour.)

Abstract:【Objective】As an important auxin primary response factor,Aux/IAA is involved in all stages of plant growth and development. Auxin sensing and signal transduction are synergistic effects of various components, in which auxin/indole-3-acetic acid (Aux/IAA) protein plays a key role. Despite their physiological importance have identified in various woody plants, the functions of Aux/IAA genes in longan are still not clear. The purpose of this study was to identify Aux/IAA family genes in longan and analyze their sequences and expression, so as to understand the roles of Aux/IAA family genes in longan especially during flower bud differentiation.【 Methods】Aux/IAA gene was identified from the longan genome using Arabidopsis Aux/IAA gene as reference, and the domain of the Aux/IAA gene family was further confirmed by the Hidden Markov Model (HMM). With the help of ExPASy, MEGA 6.0, DNA-MAN，Cytoscape tools and MEME, PlantCARE, STRING and iTOL online website, the bioinformatics of the gene family was analyzed. Their nucleotide sequence characteristics (including the chromosomal location, homology identification, gene structure, the promoter cis element) and the characteristics of the encoding protein (including physical and chemical properties of protein, conservative structure domain and motif, Protein-Protein Interaction network and phylogenetic analysis) were studied using the software. The expression of Aux/IAA gene was analyzed based on RNA-Seq database. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to study the expression of Aux/IAAgene during the process of flower bud differentiation and treated with exogenous IAA in longan.【Results】18 Aux/IAA genes in longan were identified using reference genomic data. Chromosomal location analysis showed that 18 Aux/IAA genes were concentrated in the scaffold range from number 1 to 3101. 18 Aux/IAA genes were named DlIAA1-DlIAA18 according to their locations. The physicochemical properties of Aux/IAA (DlIAA) proteins in longan were that the isoelectric point pI ranged from 5.17 (D1IAA4) to 9.31 (D1IAA17), and the molecular weight ranged from 19 562.08 Da (DlIAA14) to 55 478.99 Da (DlIAA17). Except DlIAA5, the other DlIAA proteins were unstable hydrophilic pro- teins. According to the phylogenetic relationship, evolutionary tree was divided into four classes and nine sub-tribes, in which longan had high homologous with the apple, while had distant relatives withArabidopsis thaliana. Genetic structure and multiple comparison analysis indicated that longan Aux/IAAgene families had more than one conservative amino acid sites, and contained different numbers of exons (ranged from 2 to 6). Among different branches, introns and exons varied in length. DlIAA-2, -8, -12, -18 and DlIAA-1, -2, -3, -4, -8, -14 lacked the conserved motif of Domain I (motif5), DlIAA-1, -2, -3, -4, -8 and -14 lacked the conserved motif of Domain IV-2 (motif4). A lot of cis-elements were found by the analysis of DlIAA promoters, including cis-elements related to flowering in- duction, such as low temperature response motif, circadian rhythm control base sequence motif and light response. The motifs associated with hormone response also were found, such as auxin, methyl jas- monic acid, gibberellin, abscisic acid and salicylic acid. The presence of these cis-regulatory elements indicated that hormones had potential regulatory effects on Aux/IAA genes. In the Protein-Protein Inter- action network, a total of 20 hinge genes and 72 network lines were identified, and all the other DlIAA proteins exhibited 70% high reliability except DlIAA-2, -3, -4, -6, -9, -12, -13 and -14, which suggested that proteins of the same gene family would also have parallel functions. FPKM values of 18 DlIAAsgenes in 9 different tissues of longan showed that some of the DlIAA genes were tissue-specific, nearly half of DlIAAs (DlIAA-3, -5, -7, -8, -11, -13, -14) had low expression in each tissue, while the differenc- es of DlIAA-7, -8, -13 among tissues were the least. Through the analysis of three transcriptome data of the flower buds in different longan cultivars, five genes (DlIAA-2, -4, -7, -15, -17) were found to have low expression levels during the flower bud differentiation of common longan cultivars and in the graft- ing newly-sprouting buds of Sijimi. Further qRT-PCR analysis showed that the expression levels ofDlIAA2 and DlIAA17 were significantly decreased in longan buds in November and December, which was the phase of physiological differentiation of longan flowering, suggesting that these genes might play a role in flowering induction. Treated with exogenous IAA, the expression levels of DlIAA-7, -15weresignificantlysuppressedunder the 100 μmol·L-1 IAAtreatment,whiletheexpressionlevelsofall five candidate genes increased significantly under 500 μmol·L-1 IAA concentration.【Conclusion】We identified 18 Aux/IAA members from longan genome, and found that DlIAA2 and DlIAA17 might play a key role in longan flower bud differentiation. Our results would provide comprehensive information on the genetic structure, phylogenetic relationships and expression profiles of Aux/IAA families in longan,and offer a new insight into the complexity of IAAs regulation at transcriptional level. It may be helpful to develop breeding strategies to promote the development of flowering in longan.