Analysis of the molecular evolution characteristics of miR403 and its expression pattern during early somatic embryogenesis in Dimocarpus longan

Abstract: 【Objective】MiR403, a miRNA family unique to dicotyledonous plants, plays an important role in plant disease resistance, growth and development. Previous studies showed that miR403 guided cleavage AGO2 mRNA at transcription level, which played an significant role in defensing virus. In addition, miR403 could response to stress and express specifically in different organization. Besides, when over-expressed miR403, tomato showed slow growth, dwarfism, delayed flowering, and poor fertility. Dimocarpous longan, a member of the Sapindaceae family, was an important economic fruit tree in the subtropics. According to the previous researches, miRNA is important to longan somatic embryogenesis (SE) . However, plants miR403 evolutionary characteristics and its function in plants somatic embryos have not been reported yet. Therefore, we investigated the molecular evolution characteristics of plant miR403 and its expression profile during longan early SE.【Methods】Plant miR403 matures (miRNA) and precursors (pre-miRNA) sequences were obtained from longan transcriptome database and miRBase database. The secondary structure analysis of plant pre-miR403 sequences were performed by Mfold online software. The phylogenetic tree of plant miR403 and pre-miR403 were performed by MEGA 7.0 through NJ method (Neighbor-joining) with 1000 bootstrap. In addition, the sequences multiple alignment were carried out by iTOL and WebLogo softwares. RLM-RACE (RNA ligase-mediated amplification of cDNA ends) was used to obtain longan miR403 5'c DNA (coding DNA) end sequence. The cDNA of RLM-RACE was synthesized using Gene Racer kit. DNAMAN 6.0 was used to design the primers of RLM-RACE. Based on the longan miR403 transcription start site (TSS) , the promoter sequence was extracted of 2 800 bp upstream from the TSS. The promoter of athmiR403 was collected from NCBI according to the pri-ath-miR403 (primary-ath-miR403) loading in NCBI (https://www.ncbi.nlm.nih.gov/) . Then, the TSS of citrus, soybean and grape miR403 members were predicted by BDGP (http://www.fruitfly.org/seq＿tools/promoter.html) and the TSSP (www.softbreey.com) software and promoter sequences were extracted form their genome. Next, five plant miR403 promoters cis-acting elements were predicted using PlantCARE database. Finally, the expression patterns of miR403 and pre-miR403 during longan early SE were verified by SYBR Prumix EX TaqTMII kit, Tip-mix kit and Roche LightCycler 480 real-time fluorescence quantitative PCR. The expression of miR403 was corrected by U6 SnRNA, miR408-5 p1 and ath5.8 S*, and the expression of premiR403 was corrected by FSD, EF-1α and EIF-4α. The expressions profiles of miR403 and premiR403 were obtained by 2-∆Ctmethod. The statistical analysis miR403 and pre-miR403 relative expression were performed through SPSS19.0 software.【Results】In this study, phylogenetic tree of 36 premiR403 s and 42 miR403 s sequences from longan and other 17 species showed that plant miR403 was a highly conserved miRNA family, and there was no significant difference in the number of miR403 among different species. MiR403 was mainly derived from the 3-terminal arm of pre-miR403, and only a few was derived from the 5-terminal arm of pre-miR403. The minimum folding free energy of plant pre-miR403 s was from-35.70 kal·mol-1 to-55.50 kal·mol-1. After amplifying the 5'end c DNA sequence of longan miR403 by RLM-RACE technology, a 154 bp cDNA fragment without 3'end sequence was obtained, and it was confirmed that the first base A was the TSS. The TSS of soybean, citrus and grape were predicted by the BDGP and the TSSP softwares. Then, we extracted MIR403 promoter from longan, Arabidopsis thaliana, soybean, citrus and grape genome databases for analyzing the csiacting elements. The results showed that the promoters had a large number of conserved elements such as light responsiveness elements, hormone responsiveness elements and stress responsiveness elements, implying that the regulatory factors of miR403 was common in different species. qPCR analysis showed that the expression levels of miR403 and pre-miR403 was significant decreased in longan early SE, suggesting that down-regulation of miR403 and pre-miR403 would be possible to mediate longan early SE.【Conclusion】Our researches showed that plant miR403 s was highly conserved. In addition, the down regulation of pre-miR403 and miR403 might be promotive to longan early SE.