Application Of MiPEPs In Somatic Embryogenesis Of Dimocarpus Longan Lour

Dimocarpus longan Lour.is a unique kind of fruit tree found in southern tropical areas of China.The fruit size,fruiting rate,fruit quality and harvest is dependent on the status of its embryonic development.Longan somatic embryogenesis system can be used as an important alternative model system to solve the early heterosis of longan early embryos.This constraint seriously hindered the study of in vivo embryonic development.Therefore longan somatic embryogenesis system could be used as an important alternative system.At present,the plant polypeptides encoded by mRNA as a new fertilizer can significantly increase yields and improve quality in agricultural production.Studies have shown that miPEP encoded by pri-miRNA has a significant effect on the expression and morphogenesis of plant-associated miRNAs.There is however,no published research in this aspect of study in longan.With a view to lay the foundation for the application of longan in production.The main results were as follows:1 Obtaining the pri-miRNA full length sequences and bioinformatics analysis of longanUsing RLM-RACE technique from 16 miRNAs,8 articles of 3'UTR and 10 articles of 5 ' UTR were obtained by GeneRACE reverse transcription kit.3 articles of 3 ' UTR and 5 articles of 5' UTR were obtained by SMARTTM RACE kit.They were then cloned together to obtain pri-miRNA full-length sequence of 10.It was found that the above 18 pri-miRNAs transcription start site quantity and differences were no less than one.It was found that each of the 18 pri-miRNA promoters contained a hormone-responsive cis-acting element,which varied in numbers and categories.All of them contained a variety of hormonal cis-acting elements.A total of 18 pri-miRNA promoter analyzes were performed on 16 pri-miRNA,pri-miR160 and pri-miRNA167.pri-miR160 and pri-miRNA 167 were obtained from GenBank.It was found that each of the 18 pri-miRNA promoters contained a hormone-responsive cis-acting element,which varied in numbers and categories.All of them contained a variety of hormonal cis-acting elements.However gibberellic acid and methyl jasmonic acid cis elements were common to all.According to the above findings,the 18 pri-miRNAs co-control the expression of genes by multiple response elements.2 Analysis of the expression of 19 pre-miRNA in longan miRNAUpon analysis of the expression patterns of 19 pre-miRNAs in longan EC,IcpEC,CpECGE and GE at four different developmental stages;the expression of 17 out of the 19 pre-miRNAs in IcpEC and CpECGE were detected however not high.Furthermore,pre-miR156-scaffold 29 and pre-miR156e*-scaffold 3 did not detect the expression.pre-miR 168a-scaffold2621,pre-miR2118a-scaffold26,pre-miR70-scaffold1266 and pre-miR396a-scaffold38 had higher relative expression levels in the embryogenic callus stage of the longan.The relative expression of 19 pre-miRNAs was not high in IcpEC;The expression of 16 pre-miRNAs were low except pre-miR156-scaffold29 and pre-miR395a-scaffold6 in the stage of CpECGE.The expression of pre-miR168a-scaffold2621,pre-miR2118a-scaffold26,pre-miR170-scaffoldl266 and pre-miR396-scaffold38 were higher in the stage of EC.Comparison of 19 pre-miRNAs in the 'Honghezi' longan different organs of the expression pattern.The results showed that the expression were higher in male flowers exceptpre-miR156-scaffold29,pre-miR156e*-scaffold38 and pre-miR398b-scaffold58 which were not detected among the 19 pre-miRNA.The expression of pre-miRNAs has a large regulatory effect on the growth and development of flowers,and there are differences in different pre-miRNAs in other organs.Analyzing 18 pri-miRNA promoters revealed that they had hormonal cis-acting elements.The analysis of 18 pri-miRNA promoters revealed that they all had hormonal cis-acting elements and there were also differences in the effect of the pre-miRNA response.In order to explore the respective expression patterns of the 19 pre-miRNAs,0.5 mg/L 2,4-D,2.0 mg/L gibberellin,1.0 mg/L indole-3-acetic acid,25 ?mol/L salicylic acid,10 ?mol/L abscisic acid and 25 ?mol/L jasmonic acid were used as treatment on longan callus for 24 h.The results showed that pre-miR319-scaffold517 had a significant expression with auxin 2,4-D,indole-3-acetic acid and gibberellin treatments.The expression of pre-miR168a-scaffold2621 after the treatment with auxin 2,4-D and indole acetic acid were significantly enhanced.The 2,4-D showed a significant inhibitory effect on pre-miR482a-scaffold26.The expression of pre-miR168a-scaffold2621,pre-miR 170-scaffold1266 and pre-miR319-scaffold517 were promoted by indoleacetic acid treatment.The expression of pre-miR398b-scaffold58 was inhibited by salicylic acid treatment.Under gibberellin treatment,the expression of pre-miR 160-scaffold209 and pre-miR171f-scaffold537 were significantly inhibited,whiles the expressions of pre-miR319-scaffold517,pre-miR394a-scaffold3884,pre-miR395a-scaffold6 and pre-miR396a-scaffold38 were very effective under same treatment.The expression of pre-miR395a-scaffold6 also was significantly inhibited by methyl jasmonate treatment.3 Study on miPEP in somatic embryogenesis of longan15 ?mol/L of miPEP319-1,miPEP319-2 and miPEP319-3 from the different transcription initiation sites of pri-miR319 were used as treatment on longan callus for 3 days.The results showed that miPEP319-2 had obvious inhibitory effect on the expression of miR319 family members while miPEP319-1 and miPEP319-3 had less effect.To investigate the effect of different concentrations of miPEP319-2 on the expression of miR319 family members in longan somatic embryos.The results showed that 0.4 ?mol/L miPEP319-2 promoted the expression of miR319 family members however the effect of miR319c was the most obvious.Exploring the effects of treatment time on the expression of miR319 family members.The results showed that the expression of miR319 family was significantly different within 24 h,however its effect was slow.To explore the effect of different amino acid sequences on the expression.It was discovered that both of 0.4 ?mol/L miPEP319-2 and scrambled miPEP319-2can promote the expression of different members of the miR319 family,but their degrees of promotion were different.4 Application of miPEP319 in somatic embryogenesis of longanIn order to explore the applicationof miPEP319suspension culture cycle in longan by pri-miR319,which had multiple transcription initiation sites and had a great effect on plant morphology and stress resistance.The results showed that the effect of miPEP319-2 and scrambled miPEP319-2 on miR319c were more significant in the early stage of somatic embryogenesis in longan while inhibiting the expression of longan pre-miR319a-scaffold517,pre-miR319a-scaffold1667 and pre-miR319a-scaffold1709.The miPEP319-2 had an inhibitory expression to Dlo₀22819_TCP4 and Dlo₀25379-GMMY,while the expression of Dlo₀20213_TCP4.Dlo₀26743_TCP4 and Dlo₀21198.2-GMMYB could be promoted.Scrambled miPEP319-2 in addition to Dlo₀20213_TCP4 have a promotion effect,the expression of the other four targets are inhibited.From the morphological findings after scrambled miPEP319-2 and miPEP319-2 treatment,it was observed that longan cell pellet particles became larger,and the color darker,microscopic observations showed cells to arrange more closely and also cell sizes were found to not be uniform.Effects of miPEP encoded by longan pri-miR319 at different transcription initiation sites on somatic embryogenesis in longan.The results showed that miPEP319-2 and miPEP319-3 had similar effects on miR319 family members,pre-miR319 and target.There was a difference in the effect between miPEP319-1 and them that miPEP319-1 promoted the expression of three members of the miR319 family and target of Dlo₀26743_TCP4;While inhibition of pre-miR319a-scaffold213,pre-miR319a-scaffold517,pre-miR319a-scaffoldl 667 and pre-miR319a-scaffold1709.It could be seen that miPEP encoded by pri-miR319 transcription initiation site is activeThe expression of miRNA,pre-miR319 and target expression in the early stage of somatic embryogenesis were common and different,which include miPEP319-1,miPEP319-3,miPEP319-2 and scrambled miPEP319-2 encoded by different transcription initiation sites of pri-miR319.By comparison,it was found that miPEP319-2 and miPEP319-3 had similar effects on miR319 family members,pre-miR319 and target.The effect of miPEP319-1 was different.miPEP319-1 promotes the expression of three members of the miR319 family inhibiting expression of pre-miR319a-scaffold213,pre-miR319a-scaffold517,pre-miR319a-scaffold1667 and pre-miR319a-scaffold1709.It Promotes the expression of target Dlo₀26743_TCP4.It could be seen that miPEP encoded by pri-miR319 transcription initiation site is active,and the effect of different transcription initiation sites and amino acid sequences on the development of longan somatic embryos were different.To investigate the effect of miPEP on somatic embryogenesis of longan,it was based on the effect of miPEP319.The different miPEPs can be used by encoded in the form of fertilizer andlarge quantities used in production to solve the problems in the production process efficiently and conveniently of longan.