Studies On The Mechanism Of MiR159/319 Family During Early Somatic Embryogenesis In Dimocarpus Longan Lour.

Longan(Dimocarpus longan Lour.)is a kind of evergreen woody fruit tree belonging to Succulaceae.It has high nutritional value and medicinal value.Embryonic development is an important factor affecting fruit yield and quality.However,there are many factors influencing the embryonic development of longan.Longan somatic embryogenesis and zygotic embryo development are almost similar,so longan somatic embryo occurrence is often used as a substitute for the study of longan embryo development.The miR159/319 family is a unique and conserved class of miRNAs in plants.A large number of studies has shown that miR159 and miR319 were involved in the regulation of plant growth,trophic phase transition,and stress response,whereas the miR159/319 family the mechanism of regulation in longan was not clear.In this study,the longan somatic embryogenesis system was used to perform sequence analysis,primary cloning,promoter isolation,target genes and eTMs prediction and functional verification of the miR159/319 involved in the regulation in order to deepen miR159/319 in longan.The study of regulatory mechanisms and functions during embryonic development provides new clues.The main results were as follows:1 Screening,cloning and bioinformatics analysis of longan miR159/319 family membersLongan miRNA library and transcriptome database were used to screen 10 members respectively of miR159/319 mature and precursor.Bioinformatics analysis revealed that the homology of miR159/319 family members in longan was related to the plant attributes and genesis sites.The pre-miR159/319 has a variety of ways to generate matures,in which pre-miR159a can generate miR159a-3p and miR159a-5p,and pre-miR319a can generate miR319a-3p.1,miR319a-5p.1 and miR319a-5p.2.The GeneRACE and SMARTer(?)RACE kits were used to reverse transcription,the full-length of pri-miR159a/319a were cloned that were 542 nt and 797 nt.Sequence alignment showed that precursor sequences were mainly generated in conserved regions of primary sequence,and both them didn’t contain introns.2 Cloning and cis-acting elements analysis of promoters of miR159/319 family members in longanThe 3000nt sequence upstream of pre-miR159a/319a was extracted using the longan genome,and the promoter sequences of 2771 nt and 2540 nt were cloned from the longan genome using PCR.The prediction of CpG islands showed that there were not predicted CpG islands in the miR159a/319a promoters of longan.Analysis of cis-acting elements revealed that the longan miR159a/319a promoter contains not only a large number of TATA-box and CAAT-box,but also light-responsive elements,stress-responsive elements,hormone-responsive elements,endosperm-expression response elements,growth-response elements and some of the functionally unknown components.However,only the miR159a promoter contained "TGA-element" auxin response elements that influence the "dominant role" of somatic embryogenesis in longan.3 Prediction and validation analysis of target genes and endogenous trapping targets in longan miR159/319 familyThe results of psRNAtarget prediction showed that there were 19 and 13 target genes respectively of miR159/319 families,and it was showed that a phenomenon that the same miRNA regulated multiple target genes and multiple miRNAs regulated the same target gene.The predicted binding sites were all located in the gene ORF region.The improved RLM-RACE method was used to verify the cleavage relationship between miRl 59a-3p with MYB33-like and GAMYB-like,and miR156a with SPL6-like、SPL9-like and SPL16-like.There were predicted 31 and 25 potential eTMs of miR159 and miR319 families by TAPIR,which only eTM31251 and eTM14439 were lncRNA genes,and the others were encoded protein genes.MiR159a-3p and miR319a-3p.1 were over-expressed using miRNA maturation mimics.The results showed that miR159a-3p has potential negative regulatory relationships with eTM1866 and eTM31251.4 Analysis the expression patterns of miR159a/319a in the early stages of somatic embryogenesis and related hormone treatment in longanIn the early stage of somatic embryogenesis in longan,it was found that there was no significant change about quality and morphology within 0-3 days,and the proliferation rate of cells was accelerated on the 6 th day.There were a large number of early spherical structures that protruded from the cell mass,and typical spherical embryos could be formed on the 12th day.qPCR results showed that miR159a/319a was significantly up-regulated from 0 to 12 days,and its target genes and eTMs were down-regulated.It was speculated that the miR159a/319a regulatory mechanism could promote early somatic embryogenesis in longan.The results of different embryogenic cultures of longan indicated that miR159a/319a was up-regulated with the increase in the compactness of longan somatic embryos,again confirming that miR159a/319a can promote the early somatic embryogenesis of longan.Longan EC was used to induce different hormones.The results showed that miR159a inhibited the expression in high concentrations of 2,4-D,while low concentrations promoted the expression of miR159a.There was no obvious change in the expression of miR159a induced by ABA and GA3,and showed "W"-type expression trend in ethylene induction.The expression trend of the type showed that longan miR319a was down-regulated in both 2,4-D and ABA treatments with increasing concentrations.No significant changes were observed in GA3 induction and showed an "N"-type expression trend in ethylene induction.5 Functional verification of miR159a in the early somatic embryogenesis of longanmiR159a mimics miR159a-3p-agomir and miR159a-3p-antagomir were synthesized,and then transported into longan embryogenic callus.The results showed that super-expression of miR159a-3p can suppress the formation of globular embryos and cause cell lysis.The over-expression of miR159a-3p can promote the formation of globular embryos,while miR159a-3p inhibits the expression of globular embryos in longan.The qPCR results showed that miR159a-3p,SPL6-like,SPL16-like,and SPL9-like were significantly higher in the treatment group that promoted globular embryogenesis than those in the CK group,and MYB33-like,GAMYB-like and miR156a were significantly lower than those in the CK group.In contrast to the globular embryogenesis treatment group,it was further verified that the miR159a-3p regulatory network played an important role in the early somatic embryogenesis of longan.Through the combination of miR159a-3p and miR156a over-expression and inhibition expression,it was found that miR159a-3p over-expression treatment can inhibit the expression of miR156a,while miR159a-3p inhibition expression treatment promote the expression of miR156.The expression trends of MYB33-like,GAMYB-like and miR156 were nearly identical in this treatment,which verified the potential regulatory relationship of longan miR159a-3p to miR156a.