Analysis of the molecular evolution characteristics and the expression of miR395 at the early stage of somatic embryogenesis in Dimocarpus longan

Abstract:【Objective】MiR395 is an ancient and highly conserved family of miRNAs, which plays an important role in nutrient absorption and assimilation, and is strongly induced by sulfur deficiency stress. Previous researches showed that miR395 guided cleavage APS genes and Sultr2;1 at post-transcriptional level, which played an important role in resisting abiotic stress, such as heavy metal, plant disease, chilling injury and sulfur nutrition. MiR395 family generally comprises multiple miR395 members in plants, which may exhibit functional redundancy and specificity. Longan (Dimocarpus longan Lour.) is a kind of subtropical and tropical fruit tree is economically valuable in Southeast Asia. In longan, embryo plays an important role, and the development of embryos is closely related to seed size,fruit quality and yield. Studies have shown that miRNAs are important to longan somatic embryogenesis(SE). However, miR395 evolutionary characteristics and its function of plants somatic embryos had not been reported yet. Therefore, we studied the molecular evolution characteristics of miR395 and its expression at the early SE in longan.【Methods】The matures and precursors of miR395 family members were extracted from longan miRNA library, transcriptional database and miRBase database. Then we analyzed the number of all the pre-miR395 and miR395 in plants. We carried out the longan sequence alignment using DNAMAN6.0. And the phylogenetic trees of mature and precursor miR395 sequences of plants were performed by MEGA7.0 through NJ(Neighbor-joining) method with 1000 bootstrap,then we made use of the iTOL online software to beautify. In addition, their conserved sequences were analyzed by Web Logo software, Mfold online software was used to predict longan pre-miR395 secondary structures. In order to obtain the longan miR395 s' target genes, psRNATarget online software was uesed. Finally, SYBR Prumix EX TaqTMII kit, Tip-mix kit and Roche LightCycler 480 real-time quantitative PCR method were used to verify the relationship between the predicted target genes and longan miR395 a and their expression patterns at longan early SE. During longan SE, U6 snRNA was used to correct the expression of miR395 a, and DlRan3 A gene was used as the internal reference for the target genes. The relative expression levels of the miRNAs and target genes were determined using the 2-∆ ∆ Ct method. The statistical analyeis were performed using SPSS 23.0 software.【Results】In this study, the analytical alignment results indicated that longan miR395 family contained 5 precursors and 2 mature members. Mfold analysis showed that longan pre-miR395 sequences could form a typical stable stem loop structure, but there was no significant correlation between sequence length and minimum folding free energy. The minimal folding free energies of longan pre-miR395 s ranged from-44.70 kal·mol-1 to-64.80 kal · mol-1. The phylogenetic trees of pre-miR395 s in longan and other fruit plants suggested that longan would be more closely related to Citrus sinensis, Vitis vinifera and Malus domestica. Then the phylogenetic tree of 258 pre-miR395 s and 307 miR395 s sequences from longan and other 37 species showed that miR395 was mainly derived from the 3-terminal arm of pre-miR395, and a few were derived from the 5-terminal arm of pre-miR395. There might be a loss of miRNAs similar to the case of miR156 and miR403. Multiple sequence alignment of these pre-miR395 members showed that the regions producing mature miRNA were highly conserved. Meanwhile, the evolutionary speeds of different members of the same species were different, and as the directions of evolution were different, the functions would be not alike. In addition, the evolution of different members of different species might be very close, so their functions might be very similar, indicating that their functions were also conservative. Target gene prediction of longan miR395 family suggested that there would be 6 target genes, including leucine-rich repeat receptor protein kinase LRR-RLKs, ATP sulfated APS1, and Protein phosphatase PP2 C, the prerRNA-processing protein TSR1, Leucine-rich repeat receptor protein kinase LRR-RLKs, Ketoester CoA thiolase KAT2, the EH domain protein EHD1, resoectively. In addition, according to the research of our group Zeng Youjing, longan MIR395 promoter contained auxin(2,4-D), gibberellic acid(GA3), salicylic acid(SA), methyl jasmonate(MeJA), abscisic acid(ABA) and ethylene(Eth) cis-acting elements, moreover longan pre-miR395 a responded strongly to GA3, ABA and MeJA. After 24 hours of treatment with these hormones, the relative expression level was significantly increased, suggesting that longan miR395 might play a special role in the metabolic process. Then our qRT-PCR analysis showed that the longan miR395 a was significantlyand negatively correlated with APS1, which was consistent with the results in Arabidopsis thaliana, Oryza sativa, Zea mays and other plants. The qRT-PCR assay indicated that the expression levels of miR395 a increased significantly at early SE in longan.【Conclusion】In this study, the number of longan miR395 members was discussed. In addition, our researches showed that miR395 s were highly conserved and species-specific. Simultaneously, the up-regulated expression of longan miR395 a might promote the morphogenesis of longan globular embryos.