Cloning and expression analysis of AGO10 gene from embryogenic callus in Dimocarpus longan Lour.

Abstract: 【Objective】Argonaute family proteins are highly conserved basic proteins that widely exist in the biological community and distribute in different individuals. As the core component of RNA-induced silencing complex (RISC) , the Argonaute (AGO) family proteins play a crucial role in RNA interference (RNAi) . In plants, the combination of AGO protein and small RNA participates in maintaining the stability of the gene, regulating the development of tissue, embryonic development and cell differentiation as well as responsing to hormones and stress. The objective of the current study is to discover the effect of argonaute gene on the embryogenic callus and different tissues of‘Honghezi'longan and the response to various hormones and abiotic stresses.【Methods】Based on the database of longan transcriptome, the cDNA sequences of argonaute gene were isolated from longan embryogenic callus (cultivar:‘Honghezi') by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) . Based on the unigene sequence (CL2736.Contig4) of the DlAGO10, two pairs of 5'RACE primers (universal primer UPM-Long and DlAGO10-5 RACE1, universal primer UPM-Short and DlAGO10-5 RACE2) were designed to amplify the 5'-terminal nucleotide sequence of DlAGO10, which was composed of 439 bp, and two pairs of 3 RACE primers (universal primer UPM-Long and DlAGO10-3 RACE1, universal primer UPM-Short and DlAGO10-3 RACE2) were designed to amplify the 3'-terminal nucleotide sequences of DlAGO10, which was composed of 368 bp. After fragment assembly, the full-length cDNA sequence was obtained, which was 3 859 bp. Based on the 5'-and 3'-terminal nucleotide sequences, one pair of gene specific primers (DlAGO10-CF/DlAGO10-CR) was designed to check whether the stitching of full length is correct. Bioinformatics analysis softwares (for example NCBI, DNAMAN6.0, ExPASy, ProtParam and so on) were used to predict the biological information of DlAGO10. Real-time quantitative PCR (qPCR) assay was used to analyze the relative level of gene expression during somatic embryogenesis (SE) in different longan tissues, and in response to hormones and abiotic stresses. The process of longan SE contained five stages, including non embryogenic callus (NEC) , embryogenic callus (EC) , incomplete-compact pro-embryogenic cultures (ICpEC) , globular embryos (GE) , and cotyledon embryos (CE) . The samples of longan tissues included roots (R) , stems (S) , leaf buds (LB) , leaves (L) , floral buds (FB) , alabastrum (Al) , male flowers (MF) , female flowers (FF) , filament (F) , anther (An) , young fruits (YF) , ripe fruits (RF) , and pulps (P) . Hormones applied in the experiment included 2, 4-D at concentrations of 0, 0.5, 1.0 and 1.5 mg · L-1, KT at 0, 0.25, 0.5, 0.75, 1.0 and 2.0 mg · L-1, and salicylic acid (SA) at 0, 25, 50 and 75 and methyl jasmonte (MeJA) at 100μmol · L-1. Abiotic stress treatments included salinity (NaCl, 150 mmol · L-1) , osmotic stress (mannitol, 150 mmol · L-1) , drought (PEG4000, 10%) , and abscisic acid (ABA, 10 mmol · L-1) at different time of treatments (0, 1, 2, 4, 8, 12, 16 and 24 h) . The relative expression of DlAGO10 was calculated by reference genes (EF-1 a, eIF-4 a and FSD1 a) .【Results】After verification, the DlAGO10 was isolated and the full-length sequence was successfully cloned from embryogenic callus of‘Honghezi'longan (GeneBank accession number is MH708535) . The full-length cDNA was 3 859 bp, containing ORF sequence3 003 bp and encoding 999 amino acids. The 5'untranslated region (5'UTR) length of DlAGO10 was439 bp, and the 3'untranslated region (3'UTR) length was 368 bp, including 21 bp PloyA. Bioinformatics analysis showed that the conserved domain of DlAGO10 had the typical domain characteristics PiWi of AGO and had a transmembrane domain, and the N-terminal contained 1 strong transmembrane helice with a score of 642. The DlAGO10 protein was a hydrophilic alkaline protein, and the number of positively charged amino acids (Arg+Lys) was more than that of negative charged amino acids (Asp+Glu) , indicating that DlAGO10 was a stable protein. So the number and type of amino acids were related to the structure and function of AGO. Phylogenetic tree analysis indicated that DlAGO10 belonged to the same branch as Citrus sinensis and Citrus clementina. The qPCR results showed that DlAGO10 had higher expression in non-embryogenic callus (NEC) , cotyledon embryos (CE) , and S5 stage, while the least relative expression was found in embryogenic callus (EC) , and S2 stage of‘Honghezi'longan.In different organs of‘Honghezi'longan, qPCR results showed that DlAGO10 had the highest relative expression in female flowers (FF) , the second place was in male flowers (MF) , the third position was in the leaf buds (Lb) , and the other organs (roots, stems, leaves, floral buds, alabastrum, filament, anther, young fruits, ripe fruits and pulps) had the low relative expression. A certain concentration of 2, 4-D, KT and SA can improve the relative expression of DlAGO10. When the concentration of 2, 4-D was 0.5 mg· L-1, the relative expression of DlAGO10 was the highest, which was more than twice as much as the relative expression of the control (2, 4-D concentration was 0 mg · L-1) . When KT concentration was 0.25 mg· L-1, the relative expression of DlAGO10 was the maximum, which was 2.4 times more than the control (KT concentration at 0 mg · L-1) . When SA concentration was 50 μmol · L-1, the relative expression was the highest, while different concentrations of MeJA were not only positive regulation but also negative regulation of DlAGO10. Through a certain concentration of NaCl, mannitol, PEG4000 and ABA treatments for a period of time, the relative expression of DlAGO10 can be upregulated. However, the different kinds of abiotic stresses had the optimum treatment hours. The optimal hours of treatment with NaCl (150 mmol · L-1) , mannitol (150 mmol · L-1) , PEG4000 (10%) and ABA (10 μmol · L-1) were 4 h, 8 h, 16 h and 1 h, respectively, while the least relative expression were from the treatments of 0 h, 0 h, 24 h and 24 h. Also, the overall response trend was different under different stress conditions.【Conclusion】The results suggested that DlAGO10 might be involved in the early and late transcriptional regulation of longan embryo genesis. Furthermore, DlAGO10 might participate in floral organ development and the responses to various kinds of hormone and abiotic stresses, like auxin, cytokinin, salicylic acid and ABA stressed signal transduction pathway.