Studies On Expression And Regulation Of TIR1 And MiR393 In Somatic Embryogenesis Of Dimocarpus Longan Lour.

Dimocarpus longan Lour.is a kind of important fruit in tropical and subtropical areas of China,which has extremely important nutritional and medicinal value.The developmental state of embryo keeps closely relative to fruit quality and harvest,the research of embryo development has practical significance to improve the industry of longan.But there are a lot of negative factors in study of early embryo,when longan somatic embryogenesis could be used as a good alternative system.Auxin is irreplaceable in somatic embryogenesis,and auxin receptor TIR1 plays a key point in auxin signal transduction.In this experiment,longan somatic embryogenesis was used to study gene cloning,promoter isolation,function verification and miRNA analysis of DlTIR1.The main results were as follows:1.Gene cloning and bioinformation analysis of DlTIR1 in Dimocarpus longan Lour.In this experiment,4 high expressed DlTIR1 gene family members were cloned according to transcriptome database of longan embryogenic callus(GenBank accession number:SRA059205),which GenBank accession numbers were KR558759,KR558760,KR558761 and KR558762 respectively.The homology and similarity were low,the length of nucleotide sequence and amino acid sequence were quite different between 4 members,which indicated the significant evolution variation.Further analysis showed that,DlTIR1-1 and DlTIR1-4 were similar,DlTIR1-2 got farthest genetic distance,when DlTIR1-3 was fell in between and revealed DlTIR1-3 might play greater fully function.Bioinformation analysis showed that,the physicochemical properties,phosphorylation sites,transmembrane domains and three dimensional structures were similar in these menbers,which indicated functional similarity in some degree.But the concrete parameters were specific,especially in subcellular localization,functional site and coiled coil etc that revealed functional difference in different members.2.Expression pattern analysis of DlTIR1 by qPCR in Dimocarpus longan Lour.The expression pattern of DlTIR1-1,DITIR1-2 and DITIR1-4 in normal organs and tissues were same,they were highest expression in root,second in flower,and lowest in leaf and fruit.But the expression trend of DITIR1-3 was quite specific,which also up expressed in flower.The tissue specificity indicated DlTIR1 may regulate in root growth,in addition,DlTIR1-3 may be relative to floral organ differentiation in longan.In different stages of longan somatic embryogenesis,relative expression of 4 members were displayed as ’W’ approximately,which revealed they were important in regulation of somatic embryogenesis in Dimocarpus longan Lour..The expression patterns of DlTIR1 were highly similar in most treatments by hormone in embryogenesis calli,but still had some variation.IAA,ETH,ABA and GA3 could up regulate expression of DlTIR1 in some degree,when MeJA down regulated them obviously.DITIR1 also responded NPA,PP333,light,sucrose osmotic stress and cell senescence of longan.The results revealed that,DlTIR1 involved in growth and stress response in Dimocarpus longan Lour..It was a remarkable fact that,the expression patterns of DlTIR1-1,DlTIR1-2 and DlTIR1-4 were similar in different conditions that indicated the same function,when DlTIR1-3 was different.In a word,DlTIR1-3 had specific function except common regulatory effect in growth,defence reaction and hormone response of Dimocarpus longan Lour..3.Cloning and analysis of DITIR1-3 promoter in Dimocarpus longan Lour.In this experiment,by using Genomic Walking Kit,2909 bp of DITIR1-3 promoter was amplified,which had multiple cis-elements relative to light response,hormone response,tissue specific regulatory,stress response and some others correlated with plant growth and development,but had no CpG island(GenBank accession number KR558763).The results indicated that,DlTIR1-3 may involve in hormone signal transduction,defense reaction,embryo development and metabolism process of Dimocarpus longan Lour.,and verify the conclusion present by qPCR analysis.4.Analysis of codon bias of DlTIR1-3 in Dimocarpus longan Lour.Except promoter,codon bias of DlTIRl-3 was also studied in our experiment.The ENc value was 54.4,which indicated the expression and codon bias levels of DlTIR1-3 may be low.GC and GC3s value were 0.469 and 0.487,which were less than 0.5 and indicated they were prefer to use A+T in coding region and bias toward the synonymous codons with A and T at third codon position.In addition,the codon adaptation index was 0.173,which was much less than 1.0 and further revealed low codon bias as longan genome.The analysis of different plant indicated that,codon bias and expression level of TIR1 were low but variant in different species.Phylogenetic analysis showed that,the codon usage rule of TIR1 may be similar in different species,but also remained specific characteristic in the permanent evolution.Clustering result based on codon bias usage could reflect the specific evolution regularity of DlTIR1-3,but cluster by CDS was much closer to traditional classification.According to codon usage frequency,the yeast expression system was more suitable for heterologous expression of DlTIR1-3,and the difference of codon bias between DlTIR1-3 and model plant genomes were not significant,but Nicotiana sylvestris and Solanum lycopersicum may be the best receptors for transgenosis of this gene.5.Prediction,verification and expression analysis of miRNA that related to DlTIR1-3 in Dimocarpus longan Lour.psRNAtarget prediction showed that DlTIR1-3 was only regulated by miR393,and the possible binding site was located in 3’ region of ORF.Verificated by modified RLM-RACE showed that there were 9 possible cleavage sites,and CGA/TCC may be the most possible cleavage site of this miRNA.In somatic embryogenesis and treatments under auxin and auxin inhibitors,the expression partterns of miR393 were contrary with target gene DITIR1-3,which indicated that miR393 mediated DITIR1-3 post-transcriptional control to regulate somatic embryogenesis and auxinic metabolism of Dimocarpus longan Lour..6.Function studies of miR393 and DITIR1-3 in somatic embryogenesis of Dimocarpus longan Lour.miR393 mimics miR393-agomir and miR393 antagonists miR393-antagomir were designed and synthesized,and then transported into longan embryogenic callus.the results showed that it was necessary to use cytofectin to put oligonucleotides into callus,and the expression patterns of DlTIR1-3 was contrary with miR393 that firmly meant miR393 regulated DlTIR1-3 expression.L9(34)orthogonal array was used to optimize the key factors concentration of oligonucleotide,level of cytofectin and cell density of longan embryogenic callus.The results selected the best optimal system with oligonucleotide 1μM,cytofectin 30 μL and cell density 0.15 g in total volum of 500 μL.By this optimal system,miR393 was over expression in different after treatment,and it was highest expression in 3 d,when still over 10 times after 10 d,this result indicated this method could obviously change expression of miRNA.Based on the optimal system,miR393 agomir and miR393 antagomir were transported into longan embryogenic callus to analyse the regulation on somatic embryogenesis of miR393.The results showed that inhibition of miR393 would be benefit for multiplication and growth of somatic embryogenesis,when they were abnormal growth even dead in treatment of over expression of miR393.In addition,inhibition of miR393 would improve differentiative capacity and over expression would decrease or block its differentiation.In a word,regulation of miR393 would effect auxin signal transduction to regulate somatic embryogenesis in Dimocarpus longan Lour..On the basis of miR393 optimal system,the best advantage and concentration of antisense oligodeoxynucleotides(A-ODNs)were selected with a result of ODN751 2.5μM.In follow-up experiment,the suppression of DITIR1-3 down regulated miR393,when another DlTIR1 members and downstream gene family DIARF were also decreased expression in varying degrees,which proved that DlTIR1-3 positively regulated related genes.According to above conclusions,DlTIR1-3 inhibited expression system was used to study function of DITIR1-3 in somatic embryogenesis of Dimocarpus longan Lour..It turned out that,inhibition of DlTIR1-3 would improve growth of longan somatic embryogenesis but without significant.Microscopic inspection showed that,deficiency of DlTIR1-3 caused burst and dead of cell in a large part even if it increased total weight of them.In another part,inhibition of DlTIR1-3 slowed the progress of somatic embryogenesis in Dimocarpus longan Lour..