Genome-wide identification and expression analysis of HDAC gene family in Dimocarpus longan Lour.

Abstract:【Objective】The histone deacetylase (HDAC) family, an important part of epigenetic regula- tion, plays an important role in Histone deacetylation modification. HDAC affectes the acetylation level of related genes in the process of hormones synthesis, signal transduction through the conserved lysine modified kinase on the tail of histone N terminal, as to regulate the growth and development of plants and the response to adversity. Dimocarpus longan Lour. is a famous subtropical evergreen fruit tree with high economic and medicinal value. Its flesh is rich in nutrients. The development of the embryo seriously affectes the fruit setting rate and fruit quality of the longan. Therefore, we investigated the evo- lution characteristics of the longan HDAC gene family and its expression patterns in the somatic em- bryogenesis under different hormone treatments, in order to provide a reference for enhancing resis- tance to the stresses and promoting growth, and to built the genetic basis for molecular breeding.【Meth- ods】Firstly, the candidate HDAC genes were screened from the longan genome database by BLAST  analysis in NCBI. HMMER was used to screen the sequence with the complete conserved domain as a member of the longan HDAC genes family. Secondly, ExPASy was used to analyze the physicochemi- cal properties of HDAC, subcellular localization was performed by Wolf Psort; phylogenetic analysis was performed by MEGA 5.05; secondary structure was performed by SOPMA; cis- acting elements was analyzed by Plantcare, conserved motifs was analyzed by MEME, conserved domains was ana- lyzed by Pfam and SMART, gene structure was analyzed by the gff file. The interaction between mem- bers analyzed by STRING. Thirdly, Based on the longan transcriptome datasets, we analyzed the expres- sion levels (FPKM values) of the DlHDAC family in non-embryonic cultures and embryogenic cultures of longan, as well as in nine organs of longan. Finally, the longan embryogenic callus with good growth status was obtained and treatmented in 100 μmol · L- 1 gibberellin (GA3), abscisic acid (ABA), salicylic acid (SA) and methyl jasmonate (MeJA) respectively. Roche LightCycler 480 real-time fluorescence quantitative PCR were used to analyze the expression pattern of DlHDAC family in non-embryonic, em- bryogenic cultures and with different hormone treatments【. Results】The HDAC family, could be divid- ed into three subfamilies: RPD3/HDA1,HD2 and SIR2. Among them, the HD2 subfamily was a plant- specific histone deacetylase family that has unique evolution. All HDACs were hydrophilic proteins, and most of their isoelectric points (pI) were lower than 7.0 except for SIR2 subfamily. The subcellular localization was mainly distributed in the nucleus, in a few case it was distributed in other parts such as cytoplasm, mitochondria and endoplasmic reticulum. The number of amino acids, molecular and exon- introns were changeable in different subfamilies. The number of exons in DlSRT1-2 was the highest (15), the number of exons in DlHDA8 was the least (5); the number of introns in DlSRT1-2 was the most (14), and the number of introns in DlHDA8 was the least (4). Members of the RPD3/HDA1 sub- family contained a conserved HDAC domain, HD2 subfamily members contained both a C2H2-type zinc finger domain and a Nucleoplasmin domain, SIR2 subfamily members all contained a SIR2 do- main. Nine members of the RPD3/HDA1 subfamily contained motif 2 and motif 5; Except for DlSRT1- 1, the HD2 and SIR2 subfamilies members contained motif 13. The protein structures of RPD3/HDA1 and SIR2 subfamily were mainly α-helix and random coil, while the HD2 subfamily was mainly com- posed of random coils. The cis- acting elements of the DlHDAC promoter often contained light re- sponse, hormone response, anaerobic induction, stress response, and plant growth related elements. Pro- tein interactions showed that DlHDAC protein could interact with other family members and other pro- teins. Proteins predicted to interact with DlHDAC might be functionally related to DlHDAC. Transcrip- tome data analysis revealed that most members of the DlHDAC family showed high expression both in the ICpEC and GE stage, and were highly expressed in the fruits, seeds and roots of longan. The results of qPCR analysis showed that the expression of DlHDAC was consistent with the FPKM value, among which DlHDA6, DlSRT1-1, DlHDT1 and DlHDT3 were up-regulated in GE stage. In addition, DlH- DA8, DlHDA9, DlSRT1-1, DlSRT2, DlHDT1 and DlHDT3 were induced by abscisic acid (ABA), meth- yl jasmonate (MeJA) and gibberellic acid (GA3) and salicylic acid (SA) treatments. The expression lev- els of D1HDAC gene showed an anti-“L”pattern in the SA treatment. Except for the decrease of Dl- SRT1-1 expression, other DlHDAC genes showed obvious up-regulation in the MeJA treatment. Under the ABA treatment, the expressions of DlHDA8, DlHDA9-1, DlHDT3 and DlSRT2 reached the highest peak and then decreased gradually after the treatment for 12 h. Under GA3 treatment, the expression pat- terns of DlHDA8, DlHDT1 and DlSRT1-1 were basically consistent. With the extension of treatment time, the expression trend of DlHDA8, DlHDT1 and DlSRT1-1 decreased first and then increased, and the expression level was the lowest at 8 h of treatment. These suggested that DlHDAC would regulate the somatic embryo development of longan by regulating hormone levels.【Conclusion】This study dem- onstrated that HDAC family coexisted in the evolutionary process of conservation and specificity in longan, which might be involved in the growth and development of fruits, seeds and roots, and it might reg- ulates somatic embryogenesis of longan by responding to the expression of ABA, SA and GA in longan.