Molecular characteristics of miR171 family members and analysis of the expression pattern of miR171b regulatory targets during early somatic embryogenesis in longan

Abstract: 【Objective】MicroRNA (miRNA) is an endogenous non-coding RNA that is approximately18-24 nucleotides (nt) in length. MiR171 family was firstly discovered in Arabidopsis with the function of regulating the SCL (Scarecrow-like) gene family. Subsequently, the functions of the miR171 family in other plants were discovered, including participation in hormone signal transduction, regulation of growth and development, and morphogenesis of floral organs. In addition, miR171 could promote somatic embryo maturation and respond to drought, salt, cold and other stresses. Therefore, the analysis of miR171 family characteristics, target genes, expression patterns, and other aspects would be of importance in understanding the function of miR171. Dimocarpous longan, as an important economic fruit tree in the subtropics, was a member of the Sapindaceae family. The study on somatic embryos of longan was limited by the difficulty of overestimation hybridization and somatic embryo sampling. The es-tablishment of longan somatic embryogenesis system provided a good platform for studying longan somatic embryos. MiRNAs were involved in the growth and morphogenesis of longan somatic embryos as small molecule modulators. Therefore, it was important to understand the regulatory role of miR171 in longan. We explored the molecular characteristics of longan miR171 family and its expression patterns in longan embryogenic callus (EC) treated with 2, 4-D and GA3 and in early somatic embryogenesis in longan.【Methods】Firstly, we extracted seven precursors and nine matures of miR171 from the longan genome and transcriptome databases. The miR171 mature and precursor sequences of Arabidopsis thaliana, Citrus sinensis, Vitis vinifera, Prunus persica, and Malus domestica were downloaded from miRBase. Subsequently, the bootstrap was calculated with 1 000 replicates using MEGA 7.0 constructed phylogenetic tree by neighbor-joining method. Multiple sequence alignment was made with ITOL online program. Thirdly, the target genes of longan miR171 s were predicted against the longan transcriptome database by psRNAtarget (http://plantgrn.noble.org/psRNATarget/?function=3) with the expectation 2.5. Fourthly, the modified 5'RLM-RACE was used to verify that miR171 guided cleavage the target genes SCL family using Gene Racer kit synthesized the cDNA. The volume of the PCR amplification system was 25 µL, including 1 µL GeneRacer 5'primers, 1 µL gene-specific primers, 1 µL cDNA, 9.5 µL ddH2 O and 12.5 µL mix. The PCR products were gel-purified, cloned and sequenced. Finally, SYBR Prumix EX TaqTMII kit, Tip-mix kit and Roche LightCycler 480 real-time fluorescence quantitative PCR were used to analyze the expression patterns of the target gene SCL6 and miR171 in longan EC treated with different concentrations hormones during early somatic embryogenesis. In addition, the expression of miR171 b was corrected by U6 SnRNA, mir408-5 p1 and ath5.8 s*, and the expression of SCL6 was corrected by FSD, EF-1α and EIF-4α. The relative expressions of miR171 b and SCL6 were obtained by 2-∆Ct. The statistical analysis of miR171 b and SCL6 relative expression were performed using SPSS 19.0 software.【Results】The miR171 family was widely distributed in 47 species. The mature miR171 phylogenetic tree showed that longan miR171 was distributed on different branches, indicating that the evolution rates were different. The precursor miR171 phylogenetic tree suggested that different longan pre-miR171 members could have different relationship with different plants, indicating the evolutionary sources were complex. Target gene prediction showed that miR171 family target genes mainly included SCL6, SCL15, SCL27, transcript variant X2 and RNA-dependent RNA polymerase 1 etc. This suggested that miRNA might have a powerful regulatory capacity. Meanwhile, the four target genes with the highest scores of longan miR171, miR171 b/d/f was completely consistent. Although, there were 1-4 bases difference in the sequences of four miR171 members, they also reflected the conservatism of the miR171 family. The sequences of longan miR171 c, miR171 c*2 and miR479 were significantly different from other members of the same branch in the phylogenetic tree. Longan miR171 c, miR171 c*1 and miR171 c*2 had no target genes in the longan transcriptome database. Therefore, the authenticity of the above longan miR171 members needed to be further verified. The fragment of SCL6 was identified by RLM-RACE, indicating that the miR171 b guided cleavage SCL6 mRNA. However, the cleavage site of SCL27 was beyond binding site, suggesting that SCL27 could be regulated by other miR171 s. After different concentrations of 2, 4-D and GA3 treatment, SCL6 and miR171 b showed a classic negative regulatory pattern. With the increase of 2, 4-D concentration, the transcription level of miR171 b was generally down-regulated, and the expression level of SCL6 was generally up-regulated.The expression of miR171 b was significantly down-regulation after d treatments with different concentrations of GA3, and the expression level of SCL6 reached the highest and lowest values at 2.5 and 12.5mg· L-1 GA3, respectively. SCL6 expression patterns were similar in the 0-6 d of longan early somatic embryogenesis, miR171 b. miR171 b was significantly down-regulated and SCL6 was significantly upregulated, in the 6-12 d of longan early somatic embryogenesis, suggesting that SCL6 would play an important role in globular embryo (GE) , and miR171 b would promote SCL6 expression by reducing the accumulation of it transcripts.【Conclusion】According to the study, miR171 b of longan was conservative among species and different among members of the same species. Besides, miR171 b regulating SCL6 participated in somatic embryogenesis of longan by responding to hormones.