| Longan (Dimocarpus longan Lour.), which is widely grown in subtropical and tropicalcountries, is one of the important evergreen fruit trees with high commercial value. Developmentof longan embryo is closely related to the yield and quality of longan. The middle stage duringplant embryogenesis is a vital phase, in which formation of organs and switch from a patternformation program to a storage product accumulation program occur. It is helpful in productionand breeding of longan to unveil mechanisms involved in the regulation of development of longanembryo during this phase, which is still barely understood for difficulties in collection of materialduring this phase. SE closely resembles in vivo embryogenesis, studies on plant SE can providereferences for unveiling mechanisms involved in development of embryo. In the present study,synchronization regulation of somatic embryogenesis in longan during the middle developmentalstage was performed using embryogenic callus of longan. Synchronized somatic embryos oflongan were used for proteomic analysis of longan SE during the middle developmental stage.Some differentially expressed proteins were analyzed by mass spectrometry. Cloning of Ranfamily genes were performed based on the result of mass spectrometry analysis, and furtherstudies were carried out to explore the mechanisms involved in expression regulation of Ranfamily genes. In addition, Obg gene was isolated from somatic embryos and its transcriptionallevel during SE in longan was analyzed. The present study can provide new clews and scientificbasis for unveiling molecular mechanisms involved in SE, as well as in vivo embryogenesis, inlongan and other plants during the middle developmental stage. Our main results are as follow:1Synchronization control of longan SE in longan during the middledevelopmental stageBasal medium (MS medium containing2%sucrose,6g/L agar, pH5.8) supplementedwith different concentration of2,4-D were used for synchronization control of SE in longanat the middle developmental stages. When embryogenic calli were inoculated equably onto basalmedium containing0.03mg/L and0.06mg/L2,4-D, somatic embryos at torpedo-shaped andheart-shaped stage were obtained respectively. Synchronized somatic embryos were produced at afrequency exceeded90%. Our results indicated that synchronization of longan somaticembryos can be accomplished with basal medium supplemented with different concentrations of2,4-D, role of2,4-D at different concentrations in regulating of synchronization of longansomatic embryogenesis could be related to its influences on expression of stage-specific genes.Additionally, the state of initial materials and inoculation amount were important factors thataffect the synchronization of longan somatic embryos.2Proteomics on SE during the middle stage in longanProteomic analyses of longan SE during the middle developmental stage were performedusing synchronized somatic embryos of longan. Proteins of longan somatic embryos atglobular-shaped, torpedo-shaped and cotyledonary stages were separated through two-dimensional electrophoresis using Ettan IPGphor â…¢ and DALTsix. Comparison of two-dimensional gel electrophoresis maps of longan somatic embryos at middle developmental stagesindicated that:(1) number of protein species reduced with the development of SE in longan;(2)proteomes of longan somatic embryos changed significantly during this period, changes ofproteomes during transition from globular-shaped stage to heart-shaped stage and fromtorpedo-shaped stage to cotyledonary stage were much bigger when compared with changes ofproteomes during transition from heart-shape stage to torpedo-shaped stage. These were consistentwith switches of developmental programs occurred during this period;(3) longan somatic embryosat globular-shaped and torpedo-shaped stages share a common amount of proteins, these proteinsmay be involved in cell division events.According to comparison analyses of proteome maps of longan somatic embryos during themiddle stage of SE,76differentially expressed proteins were selected for further analysis,10ofthese proteins were identified previously during proteomics of early SE in longan and maturationof longan somatic embryos. The rest of them were subjected to mass spectrometry analysis and35of them were identified. Annotation of identified proteins indicated that:(1)18of these proteinshave catalytic activity;(2)15(33%) of them were proteins with unknown function;(3)51%ofthem were involved in metabolic pathways, including alcohol metabolic, nitrogen compoundmetabolic, catabolic, biosynthetic, pigment metabolic, macromolecule metabolic, cellularmetabolic and primary metabolic processes and oxidation reduction. These results suggested thatmetabolism changes actively during this period.(4)7of them were involved in oxidative stressfurther indicated the importance of oxidative stress during SE in longan.(5)4proteins wereidentified as keratins, which were generally considered to be existed in animals, but absent inplants.(6) Active changes of expression of2small G proteins during the middle stage of SE in longan suggested that they may play roles during SE in longan.3Cloning and bioinformatics analyses of Ran family genes from longan somaticembryos and expression analyses during SE in longan(1) Cloning of Ran family genes were carried out based on the result of mass spectrometryanalysis:â‘ 31DlRan cDNA sequences, DlRan3A-1-DlRan3A-14(JF461272-JF461282,JQ775532, JQ775533, JQ861699), DlRan3B-1-DlRan3B-9(HM773390, JF461283-JF461288,JQ775530, JQ775531), DlRan3C-1-DlRan3C-3(JF461289-JF461291), DlRan3D-1-DlRan3D-2(JF461292, JF461293), DlRan3E-1(JF461294), DlRan3F-1(JQ775527), DlRan3G-1(JQ775528),were isolated from somatic embryos of longan, these cDNA sequences could be divided into9type according to specificity of ORF sequences. These genes encode7Ran proteins in total;these Ran proteins were nearly identical, amino sequence similarity of them were98.27%.Existence of differences in synonymous codon usage between transcripts of Ran gene familyenabled Ran transcripts varing in nucleotide sequence to encode the same protein. Our resultsindicated that usage of various synonymous codons may change secondary structure of mRNA.â‘¡2genomic DNA sequences of Ran family genes (DlRan3A and DlRan3B) containing intactORFs were isolated from somatic embryos of longan. Alignment of31cDNA sequences with2genomic DNA sequences showed that only some of the cDNAs were consistent with genomicDNA DlRan3A and DlRan3B in sequences. The rest of these cDNAs could be divided into twogroups. The first half sequences of cDNAs in group one were consistent with that of DlRan3A,and the second half sequences of these cDNAs were consistent with that of DlRan3B. In thecontrary, first half sequences of cDNAs in group two were consistent with that of DlRan3B, andsecond half sequences of these cDNAs were consistent with that of DlRan3A. These resultssuggested that there may be more members of Ran family genes.â‘¢Analysis of5'UTRs and3'UTRs of Ran cDNAs indicated that:â—‹a Transcription of Ran family genes from longan somaticembryos exist starting site polymorphism;â—‹b Transcripts of Ran family genes from longansomatic embryos exist polymorphism3'UTR length. In addition,4cDNA5'ends of DlRan thatcontain a premature termination codon in ORFs were obtained.(2)Bioinformatics analysis of7Ran proteins encoded by DlRan genes were located innucleus, cytoplasm and chloroplast respectively. Predicted phosphorylation sites, motifs,conserved domains, secondary structure and three-dimensional structure of these Ran protein wereclosely similar. Homology analysis indicated that Ran proteins from longan somatic embryos were highly conserved with that of other plants, the identity was more than90%. They are also highhomologous with Ran from animals. These results suggested that Ran proteins from longansomatic embryos may have similar roles as Ran proteins from other species.(3) According to amino acid10of deduced proteins,31DlRan cDNAs were divided into twogroup,. Transcription level of N and D group DlRans family genes during SE in longan wereanalyzed by qPCR using LightCycler480. Our results indicated that expression pattern of thesetwo groups of DlRans were similar to each other. Their expression remained at relatively highlevels during early period and decreased rapidly at globular-shaped stage, then accumulated to thehighest level at heart-shaped stage and followed by decrease, it reached the lowest level atcotyledon stage and increased again at mature stage, but still at a low level. The high expressionlevel of DlRan was consistent with its roles in cell cycle. Expression patterns of DlRans attranscriptional and protein level were not completely consistent, which can be the result ofpost-transcriptional regulation.4Studies on expression regulation of Ran family genes from longan somaticembyos(1)5PTC-Ran cDNAs,:DLRan,DlRan-4,DlRan-5,DlRan-6and DlRan-7, were isolatedfrom embryogenic cultures of longan. Alignment of these PTC-Ran cDNAs and correspondingwide-type transcript DlRan3B-4with DlRan genomic DNAs indicated that these cDNAs wereconsistent with DlRan3B in sequence; our results suggested that these transcripts could beproduced through alternative splicing of DlRan3B pre-mRNA. Splicing patterns of PTC-RancDNAs and corresponding wide-type transcript DlRan3B-4were compared; our results indicatedthat PTCs located in these transcripts could be introduced by intron retention. Sequences analysisof PTC-Ran cDNAs indicated that the location of these PTCs match contexts of terminationcodons to be recognized by NMD and suggested that these PTC-Ran transcripts could be targetedby NMD.To clarify whether these PTC-Ran transcripts were NMD targets, effects of CHX onexpression level of PTC-Ran transcripts were investigated. RT-PCR analysis indicated that DLRanwas also expressed in longan embryogenic calli, but DLRan2and DLRan3were not detected inlongan embryogenic calli. Expression level of DLRan was elevated in CHX-treated longanembryogenic calli. The results proposed that DLRan could be targeted by NMD. DLRan2andDLRan3were still not detected in CHX-treated longan embryogenic calli suggested that expression of PTC-Ran transcripts could be stage-specific during SE in longan. Expression levelof DLRan,DLRan2and DLRan3in longan embryogenic calli, globular-shaped and cotyledonaryembryos were analyzed by qPCR. Our results showed that DLRan were developmentally regulatedduring SE in longan, DLRan2and DLRan3were not expressed at these stages. Analysis of totallevel of DLRan and DlRan3B-4indicated that tendency of total level was similar to that of DLRan.Regularity of DLRan suggested that it could be functional. We speculated that production ofPTC-Ran could be involved in the precisely regulating of expression level of Ran from longansomatic embryos at post-transcriptional level.(2) Prediction of miRNA binding sites locating in Ran3'UTR were carried out using miRNAdatabase (developed by this lab). Our results indicated that longer Ran3'UTRs possess morepotential miRNA binding sites. Expression patterns of Ran3'UTRs different in length wereanalyzed, our results indicated that Ran3'UTR doesn't extended with the development ofsomatic embryos. We speculated that Ran could be involved in the regulation of cell division. Inaddition,3'UTRs can be involved in regulation of Ran expression during SE in longan.5Cloning of Obg1gene from somatic embryos and analysis of its expressionduring somatic embryogenesis in longan3partial sequences of Obg1cDNA were isolated from somatic embryos in longan. Sequenceanalysis indicated that DLOBG1possesses classical OBG domain and high homologous withOBG from other plants, identities with OBG from Vitis vinifera, Ricinus communis, Maize were84%,84%and82%respectively. qPCR analysis indicated that DLObg1was developmentallyregulated during SE in longan, it was expressed at the highest level in heart-shaped andtorpedo-shaped embyos. Our results suggested that DLObg1could be involved in the formation ofcotyledon.In conclusion, highly synchronous somatic embryos at heart-shaped and torpedo-shapedstages were obtained through synchronization regulation of SE during the middle stage in longan.Changes of proteome of SE during this phase in longan were investigated and35differentiallyexpressed protein spots were identified.31Ran cDNA sequences were cloned from somaticembryos of longan, and further analyses of expression of Ran during SE in longan were performed.Several PTC-Ran cDNA sequences were isolated from longan somatic embryos. The relationshipbetween PTC-Ran transcripts, as well as3'UTR, and expression regulation of Ran during SE inlongan were studied. Furthermore, DlObg1was cloned and its expression during SE in longan was analyzed. The present study can provide some clews and scientific basis for unveiling molecularmechanisms involved in SE, as well as in vivo embryogenesis, in longan and other plants duringthe middle developmental stage. |
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