Characterization Of DlRan3A And DlRan3B Gene From Embryogenic Cultures In Dimocarpus Longan Lour

The longan tree is a tropical evergreen fruit tree in southern China and has important economic value.However,there exist a lot of problems which restrict the development of longan industry.There is a lack of variety of aborted-seed type;ripening stages are concentrated;biotic and abiotic stress influence fruit quality,pre-harvest yield and postharvest storage of longan.Ras-related nuclear protein(Ran)GTPases are widely involved in growth,disease resistance,hormone sensitivity etc.Previous study showed the involvement of longan Ran during cell divisions of longan somatic embryogenesis(SE).However,to date,little is known about expression and regulation mechanism of Ran gene in longan,especially the specific role of Ran in embryo and fruit development and its involvement in response to external environment.In the present study,5' flanking sequences of DlRan3A and DlRan3B was firstly isolated and characterized;expression profiling of DlRan3A and DlRan3B was analyzed during longan SE,zygotic embryo development,pulp development,and in longan tissues,to provide new clues for Ran' role in embryo and fruit development in woody fruit tree.Meanwhile,ectopic integration,in planta transformation,and transcriptome analysis were used to analyze the roles of DlRan3A and DlRan3B to investigate their functional specificity and further the expression and regulation mechanism of Ran.Main results are as follows:1 Cloning and bioinformatics analysis of the promoters of DlRan3A and DlRan3B from longanThe 5' flanking sequences of DlRan3A and DlRan3B were cloned by using Tail-RCR and ligation-mediated PCR,1256 bp and 1569 bp in length,from embryogenic callus(EC).Promoter core region and transcription start sites(TSSs)were analyzed,and a 147bp length of CpG island was predicted in DlRan3A promoter,by using bioinformatics methods.There were elements responsive to auxin,salicylic acid(SA),methyl jasmonate(MeJA),anaerobic induction,and defense and stress in both two promoters;in DlRan3A promoter,there were other elements responsive to wounding and pathogen;in DlRan3B promoter,there were other elements responsive to gibberellin,abscisic acid(ABA),and low temperature;there might exist functional specificity between DlRan3A and DlRan3B promoters in hormone and defense responses.2 Function verification on the promoters of DlRan3A and DlRan3B from longanA series of nested 5' and 3' deletions of the DlRan3A or DlRan3B fragments were amplified for vector construction,and Agrobacterium-mediated transient assay in tobacco were conducted to analyzed the deletions and hormone responses of DIRan3A or DlRan3B promoter.The result showed that both positive and negative functional elements exist in DlRan3A or DlRan3B promoter.8.6?M IAA(indole-3-acetic acid,IAA),75 ?M SA or 100?M MeJA induced the transcription activity of DlRan3A promoter,and 8.6 ?M IAA or 34.6 ?M GA3(gibberellin A3,GA3)induced the transcription activity of DlRan3B promoter;the auxin-responsive element in DlRan3A or DIRan3B promoter might be critical positive-regulation elements;the MeJA-responsive elements might be involved in complex activation and repression regulation because of their scattered pattern in both two promoters.In DlRan3A promoter,the elements of SA responsiveness,defense and stress responsiveness and the box S were supposed to be up-regulatory elements;in DlRan3B promoter,the elements of SA,defense and stress,and low-temperature responsiveness were supposed to be negative-regulation elements;DlRan3A and DlRan3B might involve in transcriptional control in defense reactions in a specific way.3 Expression patterns of DlRan3A and DlRan3B from longanQPCR assay was used to analyze the expression profile of DIRan3A and DlRan3B gene in longan.From the expression profiling of DlRan3A and DlRan3B in different developing stages and tissues,we found that DlRan3A and DIRan3B possessed a certain expression almost in the whole longan tree;the highest expression level of both genes was detected in the seeds(S)and pulp(P);a abundant level of DIRan3A or DlRan3B expression level was also detected in cotyledon embryos(CEs)during longan SE,as well as in young embryos of early stages during zygotic embryo development,and the expression level fell rapidly with the seed germination while increased with the swelling pulp;DIRan3A and DlRan3B might involve in more important control in embryo(seed)development and swelling pulp of longan.Longan EC was used to analyzed the expression level of DlRan3A and DIRan3B under different hormone and abiotic stress treatments;the results showed that DlRan3A and DIRan3B were both induced by a certain concentration of exogenous IAA and GA3)while suppressed by high concentrations of SA and MeJA,and induced by abiotic stress(salinity,osmotic,PEG,and ABA stress)to a certain extent.It indicated that DIRan3A and DIRan3B were widely involved in response to hormone and abiotic stress.4 Characterization of ectopic expression of DIRan3A and DIRan3B driven by 35S or and their specific promoterExpression vectors of DlRan3A or DlRan3B subcellular location was constructed and were used for transient transformation of tobacco(Nicotiana benthamiana)leaves.Expression vectors were constructed,including 35S promoter,DlRan3A promoter or DlRan3B promoter-driven DlRan3A or DlRan3B gene expression vectors,DlRan3A promoter or DIRan3B promoter-driven GUS gene expression vectors,and a RNA interference(RNAi)expression vector of DIRan3A.Subcellular location assay showed that DlRan3A and DlRan3B mainly located in the nucleus.The GUS staining of specific promoter-driven GUS gene transformed tobacco indicated that DlRan3A and DlRan3B highly expressed in tissues with active cell division,like root tips and branching roots;they were also abundant in fruits and seeds,and DIRan3A showed higher accumulation in transgenic tobacco flowers than DlRan3B.The tobacco overexpressing DIRan3A driven by its specific promoter,pCAMBIA1301-pDlRan3A(1256bp)-DlRan3A(PA_A),showed stocky plants,delayed flowering,abnormal fruits and low seed rate.It indicated that DlRan3A promoter exerted significant impact on regulating growth cycle and reproduction.The PA_A tobacco showed inapparent main root with vigorous root hair,which suggest the tight relation between DlRan3A and root hair development.RNAi transgenic tobacco(ran3a)showed postponed growth,abnormal flowers and fruits and none seeds;It indicated that DlRan3A gene play an indispensible role in plant growth.The tobacco overexpressing DlRan3A,pCAMBIA1301-35S-DlRan3A(P35S_A)and PA_A,showed stronger resistance to abiotic stress(salinity,osmotic,drought,and heat stress);by contrast,the tobacco overexpressing DlRan3B,pCAMBIA1301-35S-DlRan3B(P35S B)and pCAMBIA1301-pDlRan3B(1569bp)-DlRan3B(PB_B),showed weaker resistance to those abiotic stress;P35S_B tobacco showed weaker resistance to those abiotic stress,compared with WT and especiallyshowed hypersensitivity to salinity and heat stress;the tobacco overexpressing DlRan3A or DlRan3B both showed cold stress resistance and hypersensitivity to ABA,which suggested the key involvement of DlRan3A and DlRan3B in cold stress and ABA signaling pathway.5 Transcriptome analysis of ectopic expression of DlRan3A and DlRan3B driven by 35S or and their specific promoterTranscriptome sequencing was used to analyze the transgenic tobacco(N.bentharniana)plants overexpressing DIRan3A or DlRan3B driven by 35S or and their specific promoter.As a result,longan Ran might be involved in complex activation and repression regulation in auxin signaling pathway because of the up-regulated and down-regulated DEGs involving in auxin signaling in plants overexpressing DlRan3A or DIRan3B.The up-regulated a DEGs involving in ABA transport and phenotype under ABA stress suggested that overexpressing longan Ran might accelerate plant response to exogenous ABA.Brassinosteroid(BR)-upregulated genes were significantly up-regulated,and the gene which involves in the negative regulation of BR biosynthesis was down-regulated;these expression pattern suggested that there might be more BR content and enhanced BR output in transgenic tobacco;most cell wall proteins were up-regulated,which suggested that DIRan3A and DlRan3B might be involved in regulating accumulation of cell wall substances,which influenced longan growth and development,especially longan early embryo and pulp development.The transgenic tobacco plants overexpressing longan Ran were significantly enriched in pathways of carbohydrate metabolic process and alanine,aspartate and glutamate metabolism;Besides the GO significantly enriched pathway mentioned above,there was a oxidoreductase activity,acting on peroxide as acceptor pathway in P35S_A;and in PA_A,there were structural constituent of cell wall,plant-type cell wall organization or biogenesis,cellulose synthase activity,cellulose biosynthetic process,cellulose metabolic process,glucan metabolic process,external encapsulating structure organization,etc.three extensin-like genes,with Pollen Ole e 1 domain,were up-regulated DEGs in PA_A(compared to P35S_A.),and down-regulated DEGs in PB_B(compared to P35S_B),and were both significantly enriched in pathways of structural constituent of cell wall and plant-type cell wall organization or biogenesis;combined with the phenotypes,it indicated that specific promoter-driven DlRan3A overexpression exerted significant impact on increasing levels of some extensin-like genes,which influenced root hair,fruit and seed developments;it suggested that DlRan3A and DlRan3B promoter might play specific regulating role in cell wall organization or biogenesis in longan tissues.In transgenic tobacco plants overexpressing longan Ran,there were a large amount of defense-related DEGs encoding peroxidase(PER),aquaporin(TIP),asparagine synthetase(AS),glutathione S-transferase(GST),heat shock protein(HSP)and its chaperone protein dnaJ(DNAJ),etc;there were a large amount of defense-related DEGs encoding transcription facters(TFs)including MYB,NAC,WRKY,ERF,GRAS,and bHLH families and so on;it suggested the tight relation between longan Ran and defense reaction.Compared to plants overexpressing DlRan3B,those overexpressing DlRan3A showed stronger resistance to abiotic stress(salinity,osmotic,drought,and heat stress),which might be partially attributed to the significant up-regulated defense-related genes in P35S_A and PA_A,such as PER,GST,OLP,TI,PPO and BG;the reason might also lie in the significant down-regulated defense-related TFs genes in P35S_B and PB_B,mainly including ERF,WRKY,bHLH,C3H,C2H2 and GRAS.6 Characterization of DlRan3A and DlRan3B in in-planta transformed longanIn the in-planta transformed longan overexpressing DlRan3A or DlRan3B,the genes of CESA6,EXLA2,and EXT3 were significantly up-regulated,which were related to structural constituent of cell wall and plant-type cell wall organization or biogenesis;meanwile,the defense-related MYB59,DIPPOl,and TIP1-1 genes were also significantly up-regulated,which further verify the fact that there were a large amount of cell wall-related DEGs and defense-related DEGs in transgenic tobacco overexpressing longan Ran,and suggested Ran' s close involvement in cell wall activities and resistance regulations.In summary,in the present study,the promoters of DlRan3A and DIRan3B were isolated and both of them possessed functional specificity in processes of hormone responses and resistance regulations;the small GTPase genes DlRan3A and DIRan3B were important and indispensable in longan growth and development,especially in longan embryo(seed)and pulp development.Longan Ran might involve in regulating auxin,cytokinin(CTK),ABA,and BR signaling pathways.Ectopic expression of longan Ran showed hypersensitivity to ABA and cold stress resistance;those overexpressing DlRan3A plants showed stronger resistance to abiotic stress and specific promoter-driven DlRan3A overexpression exerted significant impact on increasing levels of some extension-like genes,which influenced root hair,fruit and seed developments;longan Ran was closely involved in cell wall activities and resistance regulations.