Cloning And Function Analysis Of DIDCLs In Somatic Embryogenesis Of Dimocarpus Longan Lour.

Dimocarpus longan Lour.is a Sapindaceae and evergreen woody fruit tree,which is a tropical valuable species in southern China.The development of the seed(embryo)is closely related to the quality of fruit.There are increasing evidences showing that the temporal and spatial gene expression during plant somatic embryogenesis is not only controlled by gene sequences but also by epigenetic regulation.However,among epigenetic regulation,one of the most important modifications is DNA methylation,and certain degree of DNA methylation effect the development of plant embryos.Recent studies have shown that miRNAs,especially the 24 nt ImiRNA,are closely related to DNA methylation.But,to date,little is known about the mechanism of ImiRNA mediated target genes expression by DAN methylation,and the function of Dicer-like(DCL)protein in longan somatic embryogenesis(SE)and the mechanism of its response to external environment are not clear.Our previous work showed that miRNA played an important role in longan somatic embryogenesis,however,miRNA produced by the cleavage of DCL,which suggested that DCL played an important regulatory role in longan SE.Therefore,in the present work,based on the unigene sequences of transcriptome database of longan embryogenic calli,the open reading frames(ORFs)sequence of longan's DIDCLs were successfully cloned and analyzed by means of bioinformatics.Then,based on the longan genome data,the promoter sequence and cis-elements of different DIDCLs members were cloned and analyzed.Meanwhlie,the expression patterns of DIDCLs in the development stages of somatic embryos and different tissues of longan,the expression profiles responses to the biotic and abiotic stresses,as well as their expression under DNA methylation inhibitor(5-azaC,5-azacytidine)were determined by qPCR.In addition,antisense oligonucleotide silencing technology was used to study the function of DIDCLs in the early stage of somatic embryogenesis,which provides the foundation for the further research on the regulation of DIDCLs gene during longan somatic embryos.The main results are as follows:1.Gene cloning and bioinformatics analysis of DIDCLs in longanThere are four members of DCL family genes,i.e.DIDCL1,DIDCL2,DlDCL3 and DIDCL4,and different DIDCLs genes were differentially expressed at somatic embryogenesis stage based on the FPKM value of Unigene.The open reading frames(ORFs)of Unigene8573,Unigene22241,Unigenel9574,and CL2379.Contig2.members with higher expression levels in FPKM were cloned and verified.GenBank accession numbers were MF001060,MF001061,MF001062,and MF001063,respectively.Sequence alignment analysis revealed that the homology and similarity between the four amino acid sequences were low,indicating that a deviation occurred during the evolution of DlDCLs.Bioinformatics analysis showed that the physical and chemical properties of the DIDCLs are mostly similar,all of them are hydrophilic and unstable proteins with transmembrane structure but without signal peptide.Meanwhile,there are also some differences:DlDCL2 is basic protein while DlDCL1,DlDCL2 and DlDCL4 are acidic proteins.Subcellular localization prediction showed that DlDCL2 was located in nucleus and chloroplast,while the other three members were located only in the nucleus.Protein domain prediction showed that DCLs were highly conserved proteins.Phylogenetic tree analysis showed that the DCLs of different species are divided into 4 clades and the homologous DCL proteins are clustered together.And the genetic relationship of DlDCLs family in longan was close to those from Citrus sinensis.2.Cloning and analysis of the DIDCLs promoter family genes in longanIn order to investigate the functions of DCL genes in longan,we isolated gene sequences of DIDCL1,DIDCL2,DlDCL3 and DIDCL4 from longan genome data.The 5' flanking sequences of DIDCLs were directly cloned by PCR.The size of four sequences of 2974,2687,2702 and 2829 bp were obtained.Analysis of Cis-acting elements on the DIDCLs family members were showed that the promoters of DIDCLs genes not only included by the cis-acting elements of TATA and CAAT box,but also had a large number of light response elements,hormone response elements,stress response elements,tissue specific regulatory elements and other cis-elements for plant growth and development.These results suggested that the transcriptional activity of the promoters of DIDCLs genes might be induced by the signals of light,phytohormones and abiotic stresses.Promoter cis-acting elements for different members DIDCLs gene family comparative analysis showed that different DlDCLs promoter contain their own specific cis-elements involved in stimuli response.Besides,some of the same cis-elements also exist in the promoters of different DlDCLs members.It suggested that the same stress can modulate the expression of plurality of DlDCLs genes,but each member on the same stress response also exhibited some differences.3.Expression patterns of DIDCLs in development stages of somatic embryos,and different tissues of longanqPCR assay was used to analyze the expression pattern of DIDCLs genes in longan.The results showed that the expression patterns of different DlDCLs in development stages of somatic embryos varied significant,but DIDCLs are relatively high in the stage of embryogenic callus(EC),which may indicate that DIDCLs have distinct but cooperative functions in the process of the development stages of somatic embryos.The expression patterns of DIDCLs in different tissues were also investigated.The results revealed that DIDCL1 and DIDCL2 had the highest expression in the leaf and followed by floral organ tissues,which suggested that DIDCL1 and DlDCL2 may participate in photosynthesis and floral organs development.4.Expression patterns of DIDCLs under hormone and abiotic treatmentExpressions of DIDCLs were analyzed in EC under abiotic stress and exogenous hormone treatment in Dimocarpus longan.The results showed that:DIDCLs were significantly up-regulated by means of 0.5 mg/L 2,4-D treament,while down-regulated with concentration of 2.0 mg/L.After MeJA,SA,GA3,and ABA treatment,DIDCLs genes were down-regulated.However,they were dramatically up-regulated under high concentration of ETH treatment.The results indicated that the DIDCLs might be regulated by several kinds of hormones.Under abiotic stress treatments,the results showed that the expression patterns of DIDCL1,DlDCL3 and DIDCL4 were all up-regulated under different light quality treatment,while DIDCL2 was significantly up-regulated under red light,blue light and mixed light treatment.Under sucrose stress treatment,DlDCL2,DIDCL3 and DIDCL4 were up-regulated when they were treated with high concentration of sucrose(6%),in the exception of DlDCL1,which was significantly increased at 0.1%sucrose concentration.During heat treatment,DIDCL1 was significantly up-regulated at 34 ?,whiles the DlDCL3 gradually decreased with rising temperature.The expression level of DIDCL2 and DIDCL4 exhibited slight variations under heat treatment.NaCl treatment induced the response of DlDCLs at different time intervals and inhibited the expression of DlDCLs at 1h.The expression patterns of DIDCLs genes response to hormones and abiotic stresses suggested that DIDCLs gene were not of a simple one to one correspondence but rather a complex response mechanism.5.Effects of 5-azaC(5-azacytidine)on longan EC and the expression of DIDCLsLongan EC was cultured in solid medium supplemented with 0,1.0,1.5,2.0 and 2.5?M 5-azaC for 20 days.The growth and cell morphology of longan EC were observed.The results showed that compared with the CK,longan EC had a significant decrease in the growth under high concentration 5-azaC treatment(1.0?M,1.5?M,2.0?M and 2.5?M),whereas the growth of the longan EC increased slightly at low concentrations of 5-azaC(0.5?M).The results of the microscopic observation showed that the EC cells in 0.5?M 5-azaC treatment were not significant different with the normal cells,while cells became abnormal at high concentrations of 5-azaC.However,5-azaC has toxic effects on cells,so the choice of the suitable concentration and appropriate treatment time is particularly important.Expression of DIDCLs were analyzed in longan EC under different concentrations of 5-azaC(0,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 and 1.0?M 5-azaC)for 24h.The results showed that the expression of DIDCL3 and DIDCL4 were inhibited under 5-azaC treatment,while DIDCL1 and DlDCL2 showed the highest expression levels at 0.3 and 0.5?M respectively,suggesting that DIDCLs was involved in the response of 5-azaC treatment.6.Effect of DlDCLs gene silencing on early somatic embryogenesisAccording to the DIDCL3 and DlDCL4 gene sequences,siRNAs were designed and synthesized,and then transported into longan EC.qPCR was used to detect RNAi silencing effects.The results showed that DlDCL3-siRNA-1904 and DlDCL4-siRNA-590 had the highest inhibitory effect.Therefore,the above siRNAs were used to undertake the effect of DIDCLs gene silencing on early somatic embryogenesis.The results showed that inhibition of the expression of DIDCL3 gene accelerated the development of somatic embryos in longan to some extent.The inhibition of DlDCL4 gene expression had a certain weakening effect on the ability of longan somatic embryo development and differentiation.However,as time went by,the treatment group and the control group gradually became more synchronized in the early stage of somatic embryo development,which inferred that inhibition of DlDCL3 or DlDCL4 gene expression,other members may replace their role in the process of somatic embryogenesis.