| Longan(Dimocarpus longan Lour.),a tropical/subtropical evergreen fruit tree within the Sapindaceae family,native to South China and Southeast Asia.Logan embryo development status was close association with the seed size,fruit-set rate,fruit production and quality.Somatic embryogenesis(SE)is a process of somatic cells that dedifferentiate to the totipotent embryonic stem cells and generate embryos in vitro.Longan SE has been widely used as a model system for studying the embryogenesis in woody plants.The transcriptomics and Proteomics analysis during the early development stages of longan SE was performed in this study,which will help to better understand the molecular mechansim during longan SE and lay a foundation for the regulation of the development of longan zygote embryo.Based on the longan EC transcriptome database,we cloned the flowering time related genes from longan EC and analyzed their expression profiles.Meanwhile,functional study of flowering time related genes and its promotor was performed by genetic transformation in Nicotiana benthamiana In addition,we had established a rapid transgenic method on longan seedling which will provides a new strategy for gene functional studies and germplasm creative of longan.1.Transcriptome analysis during early somatic embryogenesis in Dimocarpus longtan Lour.RNA-seq of longan early SE(NEC,EC,ICpEC,and GE)generated a total of 243.78 million high quality reads,81.11～82.08%of the data were mapped to the reference genome,and 49.07～57.51%of the data were mapped to the reference gene,respectively,and a total of 130,354 AS events were checked across those four stages.The cDNA libraries of NEC,EC,ICpEC and GE generated 22743,19745,21144 and 21102 expressed transcripts and 1935,1710,1816 and 1732 novel transcripts,and 2645,366,505 and 588 unique genes,respectively.10642,4180,5846,1785 genes were differentially expressed in the pairwise comparison of NEC_vs_EC,EC_vs_ICpEC,EC_vs_GE,and ICpEC_vs_GE,respectively.There were 4887 upregulation and 5755 downregulation genes in the comparison of NEC_vs_EC,2689 upregulation and 1491 downregulation genes in the comparison of EC_vs_ICpEC,3451 upregulation and 2395 downregulation genes in the comparison of EC_vs_GE,832 upregulation and 953 downregulation genes in the comparison of ICpEC_vs_GE.KEGG pathway analysis indicated that those DGEs were mainly enriched in the metabolic pathway,biosynthesis of secondary metabolites,plant-pathogen interaction,plant hormone signal transduction.In addition,a lot of DEGs were enriched in the phenylpropanoid biosynthesis,flavonoid biosynthesis,fatty acid biosynthesis,zeatin biosynthesis,tryptophan metabolic.Comparative transcriptome analysis revealed the important role of auxin and cytokinin metabolic and the hormone signal transduction during longan SE.The transcripts profiling of flavonoid and fatty acid biosynthesis related genes suggested that flavonoid were mainly accumulated in NEC,while fatty acid accumulated in early SE.In addition,the DNA replication and cell cycle related genes and the extracellular protein encoding genes LTP,CHI,GLP,AGP,EPl were differentially expressed and related to longan SE.Transcript profiling combined with qRT-PCR performed on selected genes confirmed that 27 SE molecular markers(LEC1,LlL PDF1.3,GH3.6,AGL80,PINI,BBM,WOX9,WOX2,AB13,et al.)and 28 NEC molecular markers(LEA5,CNOT3,DC2.15,PRl-1,NsLTP2,DIRl,PIPl,PIP2.1,TIP2-1,POD-P7 and POD5 et al.)were characterized as molecular markers,they were predicted to play an important role in regulation of longan SE,and they may be the key factors in determining the potential of longan SE.2.iTRAQ base proteomic analysis during early somatic embryogenesis in Dimocarpus longan Lour.Base on the iTRAQ lable method,a total of 5035 proteins were indentified during the early development stages of longan SE(NEC,EC,ICpEC,GE).COG classification indicated that those proteins were enriched in "General function prediction","Posttranslational modification,protein turnover,chaperones","Translation,ribosomal structure and biogenesis","Replication,recombination and repair","Transcription","Carbohydrate transport and metabolism","Energy production and conversion","Amino acid transport and metabolism","Signal transduction mechanisms",and "Lipid transport and metabolism".The KEGG pathway enrichment analysis showed that "Metabolic pathways" and "Biosynthesis of secondary metabolites" were the most enrich pathways,which account for 41.81%of the identified proteins.Besides,"Ribosome","RNA transport","Spliceosome","Plant-pathogen interaction","Protein processing in endoplasmic reticulum","Purine metabolism",and "Plant hormone signal transduction" also the main pathways.Furthermore,82,78,103,73,160,and 116 differentially accumulated proteins(DAPs)were identified in the pair-wise comparison of NEC_Vs_EC,NEC_vs_ICpEC,NEC_vs_GE,EC_vs_ICpEC,EC_vs_GE,and ICpEC_vs_GE,respectively.The DAPs analysis revealed that the SE-related protein LECl-like,CHI5,CHI4,AGP,EP1 were differentially expressed,and might involved in the regulation of longan early SE.During longan early SE,the carbohydrate and energy metabolism related proteins were differentially expressed,such proteins related to the glycolytic pathway,pentose-phosphate pathway,ethanolic fermation,and TCA cycle were almost significantly up-regulated in GE stage,indicated that carbohydrate and energy metabolism play an important role in the process of longan SE.Meanwhile,the auxin,ABA and GA3 signaling pathway related protein,and the stress-responsive protein such as CAT,POD,APX,GPX,GST,Hsp90,Hsp60,et al.were significantly changed during longan early SE,which also suggested to be the key regulator of logna SE.In addition,the proteins involved in lipid metabolism,protein metabolism and cytoskeletal structure were differentially accumulated during early SE,and showed a close relationship with longan early SE.To better understand the relationship and interplay between transcriptome and proteome in longan SE,the correlation analysis was performed using the transcriptome and proteome data.As a result,almost all identified(96.4～97.7%)and quantifiable(98.3～99.5%)proteins had a corresponding transcript,only a few DAPs had a corresponding transcript.There were only 147 differentially expressed proteins/transcripts with the same expression trend in the six pair-wise comparison,and their correlation coefficient r were 0.3789～0.7576.However,there were 66,differentially expressed proteins/transcripts with the opposite expression trend,392proteins differentially/genes No,and 5166 genes differentially/proteins No,which further suggested that the correlation between transcriptome and proteome of longan early SE was low.Hence,the transcriptome and proteome analysis during longan early SE play an important role in studying the molecular regulation mechainism during longan SE.According to the transcription and accumulation level of Remorin were significantly changed during longan SE,five members of Remorin family were isolated from longan EC,and their expression pattern during longan SE indicated that Remorin may be involved in the regulation of longan SE.3 Cloning and expression regulations of flowering time related genes in embryonic callus of longanBased on the transcriptome data of longan EC,43 genes related to flowering time were screened and cloned by RT-PCR and RACE.Further study of expression patterns of flowering time related genes during longan SE and ZE,at different tissues and under various hormones and stress treatments.The potential functions of these flowering time genes in longan embryogenesis and other growth and development stages of longan were revealed.3.1 Cloning and expression analysis of DIMFT and D1TFLl in longan ECTo gain insight into the molecular function of DIMFT and DlTFL1 in Dimocarpus longan Lour.,we isolated DIMFT and DlTFL1 and theirs promoter sequence from longan EC.Bioinformatic analysis indicated that the DlMFT promoter contained multi-phytohormones and light responsive regulatory elements,and multiple regulatory elements which were involved in seed-specific transcriptional regulation.Subcellular localization showed that the given the DIMFT signal localized in the nucleus,expression profiling implied that DIMFT showed significant upregulation during SE and ZE,and particular highly expressed in late or maturation stages.The accumulation of DIMFT was mainly detected in mature fruit and seed,while it was undetected in abortive seeds,and notably decreased during seed germination.DlMFT responded differentially to exogenous hormones in longan EC.Auxins,SA and MeJa suppressed its expression,however,ABA,BR showed the opposite function.Meanwhile,DlMFT differentially responded to various abiotic stresses.Our study revealed that DIMFT might be a key regulator of longan somatic and zygotic embryo development,and in seed germination,it is involved in complex plant hormones and abiotic stress signaling pathways.Bioinformatic analysis indicated that the DlTFLl promoter contained multi-phytohormones and light responsive regulatory elements.qRT-PCR implied that DITFL1 showed significant downregulation from EC to CE,but undetected in NEC stage,and late stages of ZE,showing the inhibition on early SE.The accumulation of DITFL1 was mainly detected in vegetative-bud,young-stem,flower bud,and female flower,barely expressed in other tissues,while it was undetected in abortive seeds,and notably decreased during seed germination.DITFL1 responded differentially to exogenous hormones in longan EC.ETH,KT,SA,MeJa,PP333,GA3 enhanced its expression,however,2,4-D showed the opposite function.Meanwhile,DITFL1 differentially responded to various abiotic stresses.We speculated that DlTFL1 might be involved in longan early somatic embryo development,flowering regulation,floral development,and involved in complex plant hormones and abiotic stress signaling pathways.3.2 Cloning and expression analysis of phytochrome and cptochrome in longan ECTo gain insight into the molecular function of phytochrome and cryptochrome in Dimocarpus longan Lour.,we isolated DIPhyA,DIPhyB,DlPhyC and DlCry1,DlCry2,DlCry3 from longan EC.qRT-PCR indicated that DlPhys and DICrys were differentially expressed during SE and ZE,suggested that they might be involved in longan embryogenesis.Among the tested tissues,DlPhyA and DIPhyB were mainly expressed in seeds,barely or undetected in other tissues,while DIPhyC showed high expression in anthers,female flowers,inflorescence,leaves and flower buds.Meanwhile,DlCryl,DlCry2,and DICry3 were mainly expressed in leaves,and showed high expression in leaf buds,inflorescence,flower buds,female flowers,and seeds.In addition,DIPhys and DICrys might be involved in the complex light and hormones signal transduction.3.3 Cloning and expression analysis of circadian clock related genes in longan ECTo gain insight into the expression pattern of circadian clock related genes in longan,DIELF4,DlELF4-likel,DlELF4-like4,DIEL1,DIELF3,DIELF6,DlZTL,DlKelch,DlLHY,DlFKF1,DlTOCl,DlGI,DIC09,and DlCO10 were isolated from longan EC.qRT-PCR revealed that DIELF4 family and might be involved in longan embryogenesis,flower formation and fruit development processes,and involved in the hormones signaling and stress-responsive.Meanwhile,DIEL1,DIELF3,DICO10 DlGI might be involved in longan embryogenesis and flower formation.Moreover,exogenous GA3 and BR promoted the expression of DlELI DIELF3,DlCO10,DIGI.3.4 Cloning and expression analysis of six members of GRAS family in longan ECTo gain insight into the molecular function of GRAS in longan,we isolated six members of GRAS family,DlGRAS4,DlGRAS54,DlGAI,DlGAIP-B,DISHR,DIRGL2 from longan EC.Anglicizing phylogenetic tree of GRAS in plants indicated that DlGRAS54 belonged to the PAT1 branch,DlGRAS4 belonged to the SCL branch,DlGAI,DlGAIP-B,DlRGL2 belonged to the DELLA branch,and DlSHR belonged to the SHR branch.qRT-PCR indicated that DIGRAS4 showed approximately a"W"curve,it expressed at the highest levels in globular and cotyledonary embryo stages.DlGRAS54 showed approximately an"M" curve,it expressed at the highest levels in globular and topedo embryo stages,suggesting that DlGRAS4 and DlGRAS54 played a major role at the middle and late developmental stages of longan SE.Meanwhile,DlGRAS4 and DIGRAS54 showed up-regulated under GA3 treatment showed that DlGRAS4 and DlGRAS54 made the positive response with GA3.qRT-PCR indicated that DIGAI and DIGAIP-B were differentially expressed during longan ZE.The expression of DIGAI and DIGAIP-B were up-regulated during seed germination,they might played an improtant role in seed germination.Among the tested tissues,DIGAIP-B was mainly expressed in seeds,barely or undetected in other tissues,while DlGAI showed high expression in anthers,inflorescence,leaves and female flowers,which suggested the potential role of DlGAIP-B in seed development and DIGAI in multiple-developnent processes,especially in flower formation.The expression patterns of DlGAI and DlGAIP-B under different hormons and abiotic stresses were similar,GA3 and BR significantly promoted their expression,ABA and IAA also had certain promotion effect,while SA,MeJa,NaCl,PEG4000 showed the opposite pattern,suggested that DlGAI and DlGAIP-B might be involved in hormones signaling pathway and responded to abiotic stress.3.5 Cloning and expression analysis of autonomous pathway genes and other flowering related genes in longan ECThe autonomous pathway genes DIFPA1,DlFPA2,DlFLD,the embryonic flower genes DIEMF1,DlEMF2a,DlEMF2b,the DlFLC and five members of FRl family were isolated frome longan EC.qRT-PCR showed that the autonomous pathway genes and embryonic flower genes might be involved in longan embryogenesis and showed different expression pattern among the tested tissues of longan,and they might participated in the regulation of longan growth and development.Furthermore,GA3 and BR could promoted their expression in longan EC.4 Characterization of flowering time related genes in longan EC4.1 Functional analysis of DlMFT and DlTFL1 in longan ECExpression vectors of DIMFT and DITFL1 promoter were constructed,and the DIMFT and DlTFL1 promoter-driven GUS gene highly expressed in tobacco which can be used for further study.Expression vectors of DIMFT and GFP fusion subcellular location was constructed and transient transformed into onion inner epidermal cells,subcellular location showed that DIMFT was mainly located in nucleus.The over-expression and RNA interference(RNAi)vectors of DIMFT and DlTFL1 were also constructed,and transformed into tobacco by agrobacterium tumefaciens-mediated transformation.The results showed that ectopic expression of DlMFT and mft does no effect on flowering,but ectopic expression of DlMFT inhibited the seed germination,ectopic expression of mft did the opposite function on germination.Ectopic expression of DITFL1 led to a thick green leaves,more exuberant vegetative growth,more lateral bud,delay flowering in DITFL1 transgenic tobacco,and vice versa.Suggesting that DITFL1 played an important role in flowering time regulation.4.2 Functional analysis of DISHR in longan ECThe DISHR expression vector was constructed and transformed into tobacco.Compared with the wild type tobacco,DISHR transgenic plants showed the following phenotype:the growth and development,and flowering delayed significantly,with bigger roots but fewer than WT,the roots were smooth and almost hairless,even some of the roots were markedly swollen.The stems were distorted slightly,and the leaves were smaller with distorted or fold,and epidermal hairs were thick,even part of the mesophyll owned a thick epidermal hairs.The microscopic observation of the roots revealed that the DlSHR transgenic tobacco have only a few short root hairs,have a large number of cortex and endodermis cells and showing an extremely dense collection of small cells in root.Hence,DISHR played an important role in the regulation of plant development,especially in the regulation of root,stem,leaf development,and flowering time control.qRT-PCR indicated that higher expression of DlSTHR was detected in transgenic plant with more obvious phenotype.Surprisingly,the endogenous NbSCRJ1 NbSCR2,and NbGAI showed drastically up-regulation in DISHR transgenic tobacco,we speculated DISHR might influence the development process of root and leaf by promoted the transcription level of GA-related gene GAI and root radiation maintainance gene SCR.We harvested the aboveground and underground(root)of WT and DISHR(60 day old)for transcriptome sequencing.Transcriptome analysis showed that plant hormone signal transduction pathways are the most enrichment pathways in all transgenic tobaccos.In addition,the transcription level of endogenous SCR,DELLA,Jackdaw,EXORDIUM-like family,14 KD and 36.4 KD proline-rich proteins encoding genes were regulated by DlSHR,which might played a key role in the processes of root radiation pattern,roothair development and leaf morphology.5 Establishment of a rapid transgenic method on longan seedlingIn this study,we had transformed the target gene into longan soil seedling by agrobacterium tumefaciens-mediated transformation to infect the wound,and the wound was performed by removing the apical bud or the top of the seedling.A large number of normal resistant buds were obtained through hygromycin selection,the PCR detection of target gene at different development stages indicated that the target gene had integrated into longan DNA,the further qRT-PCR analysis also detected the transcription of target gene in longan.Using this rapid transgenic method,more than 20.83%resistant buds were identified as positive transformation plant.Compared with traditional methods,this method independent of plant tissue culture,no need to culture the sterile seedlings,embryoids and suspension cell.Hence,this method has a significant role in gene functional study and germplasm creative of longan,due to its advantages of easier,rapid,efficiency,high survival and annual production. |
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