Applications Of The Endogenous Target (eTM) Regulation Of Mirnas During Somatic Embryogenesis In Dimocarpus Longan Lour.

Longan(Dimocarpus longan Lour.)is evergreen tree,and also known as ’guiyuan’.Its embryonic development affects fruit yield and quality.The development of plant embryos is a process of cell differentiation,and there are many regulatory factors in the process.MicroRNA and endogenous target mimics(eTM),as a new type of regulatory factor,play an important role in the differentiation and development of plant embryos.Plant embryos and somatic embryos are similar in morphology and molecular level,and somatic embryos are ideal experimental systems for studying plant embryonic development.eTM relies on its specific structure by complementing with the target miRNA to inhibit the expression of miRNAs.Here,miRNA and the corresponding eTM were selected from longan somatic embryo genes is to study,which provide a reference basis for regulating longan fruits quality.The main results were as follows:1 Prediction and bioinformatics analysis of eTMs in longan somatic embryos based on transcriptome data30 miRNAs,from the longan embryonic small RNA dataset,were selected for eTM prediction.A total of 155 potential eTMs were predicted according to the specific complementary structure of eTM and miRNA.Further analysis showed that these miRNA and potential eTM existed the following corresponding relationship:one eTM regulated one miRNA;one eTM simultaneous control multiple miRNA or multiple eTM regulate the same miRNA.And in the,the predicted eTM functional analysis.These potential eTMs were annotated by NCBI,Nr,Swissprot,KEGG,and GO database,and the results showed that most of eTMs are unknown protein;Followed by a variety of enzymes,such as RNA polymerase,processing enzymes,kinase enzymes,transferase enzymes,helicase enzymes,ligase enzymes,hydrolytic enzymes,ribonuclease and oxygenase enzymes;in addition,some eTMs also related secondary metabolism synthesis enzymes.2 Real-time quantitative PCR analysis of eTM and correspondingmiRNAs during early somatic embryogenesis of longanThe eTM-miRNA is paired with the target transcripts that are cleaved by their complementary miRNAs,resulting in increased expression levels of target mRNAs.Based on mentioned-above analysis,45 eTMs and corresponding 30 miRNAs were selected for expression analysis in longan callus(EC),incomplete compact pro-embryogenic cultures(ICpEC),and globular embryo(GE).Expression analysis showed that:the expression of eTM1866 and miR159a.1,eTM20206 and miR164a,eTM3727 and miR319b,eTM3727 and miR166c*,eTM8169 and miR172a,ETM19726 and miR398a,eTM1255 and miR413,eTM23730 and miR416,eTM5958 and miR419 expression showed a negative correlation trend in the development stage from EC to ICpEC.In additon,from ICpEC to GE,there was a negative correlation between eTM1866 and miR159a.1,eTM20206 and miR164a,eTM19726 and miR398a,eTM29075 and miR398bl,eTM19658 and eTM22892 and miR400,eTM23730 and miR416.These results indicated that eTM plays an important role in the regulation of miRNA expression and thus plays a role in the regulation of development of longan seed(embryo).3 Expression analysis of eTM and corresponding miRNAs under different hormone treatments in longan EC.Based on mentioned-above analysis,16 eTMs and corresponding 9 miRNAs were selected for expression analysis in longan EC,which treated with different hormone concentrations.The relative expression of miR419 and eTM5958,miR164 and eTM20206,miR159a.1 and eTM1866,miR166c*and eTM3727,miR398a and eTM19726 were measured in longan EC treated at different concentration of KT.And these eTMs and corresponding miRNAs existed a negative correlation trend in partial concentration changes of KT.The relative expression of miR164 and eTM20206,miR172a and eTM1869,miR400 and eTM19658,miR59al and eTM1866,miRl66c*and eTM3727,miR398a and eTM19726 were measured in longan EC treated at different concentration of 2,4-D.And these eTMs and corresponding miRNAs existed a negative correlation trend in partial concentration changes of 2,4-D.There was a negative correlation between the expression of miR164 and eTM20206,miR400 and eTM19658,miR166c*and eTM3727 in the partial concentration of changes in the concentration of NAP in the treatment of longan EC.The results showed that the expression of miRNA and eTM in the process of longan EC could be affected by the concentration of KT,NPA and 2,4-D hormones.KT and auxin play roles in plant growth and development,through the expression analysis of miRNA and eTM in different hormone concentrations,will lay a foundation for regulating the development of longan fruits.4 Negative correlation between eTM and corresponding miRNA validation in longan ECMiRNA silencing is a kind of synthetic oligonucleotides small molecules of miRNA by importing cells.These synthetic oligo nucleotides binds to endogenous miRNAs become into heterologous double strands,thereby reducing the inhibitory effect of miRNAs.The technique is a commonly used method for the study of miRNA function.In this study,miR398a and miR164a mimics were transferred into longan EC through liposome transfection agent.The experimental analysis confirmed that eTM19726 and miRNA398a,eTM20206 and miRNA164a have a negative regulatory role.The expression levels of eTM19726 and eTM20206 decreased when the expression of mi398a and mi164a increased.The expression levels of eTM19726 and eTM20206 increased with the decrease of expression level of miRNA398a and miRNA164a.These results further suggests that the application of eTM and miRNA in embryonic development in vitro and vivo in longan provides a new technical platform and scientific basis.