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

Longan(Dimocarpus longan Lour)is an important and special woody fruit tree in tropics and subtropics of China,which has great economic value.There are several restrictionsin the industrial production of longan such as the lack of "cokecore" varieties,over-concentration of mature periods and so on.Research on embryonic development of longan has great potential in solving these problems.Previous work indicates that DNA methylation is closely related to somatic embryogenesis.A certain degree of DNA methylation is beneficial to the normal development of plant somatic embryos,whiles DRM(Domain Rearrangement Methylase)is a key factor in the ImiRNA-mediated methylation pathway.In this view,this study cloned DIDRMs genes,bioinformatically analyzed them,analyzed their subcellular localization,cloned their promoters and analyzed their cis-regulating elements based on the transcriptome data of the longan embryogenic calli and the somatic embryogenesis system of longan.At the same time,qPCR was used to detect the expression of DIDRMs in the different tissues,somatic embryos among developmental stages,as well as longan embryogenic calli under various hormone treatments and abiotic stress.The expression of DIDRMs in vivo in longan somatic embryos were silenced using RNAi,and the observation of these somatic with microscopy will uncover the function of DIDRMs in the somatic embryogenesis,which will pave the way for further study.The results of this study are as follows:1 Cloning and Bioinformatics Analysis of Longan DIDRMsLongan transcriptome database(Genbank accession number:SRA059205)analysis and GeneBank alignment showed that there are two kinds of DRMs proteins in the embryogenic calli of longan,which are DRM1 and DRM2,and both contained complete ORF.The cDNA of longan embryogenic callus was used as a template,DRM1 and DRM2 were cloned and verified by RT-PCR.The cloned sequence was identical with the transcriptome sequence.The DRM1 gene included a 494 bp 5' UTR,a 184 bp 3' UTR,and a complete ORF.The ORF region was 1896 bp in length,and encoded 631 amino acids,and was named DIDRM1(accession number:KY990493).The DRM2 gene included a 354 bp 5'UTR,a 336 bp 3'UTR,and a complete ORF.The length of the ORF region was 2112 bp,which encoded 703 amino acids and was named DIDRM2(accession number:MF001059).Bioinformatics analysis showed that the basic physical and chemical properties of D1DRM1 and D1DRM2 were similar.They both belonged to unstable hydrophilic proteins that do not contain signal peptides.Besides,there were no transmembrane domain apart from a Dcm conserved domain,in the evolutionary tree and the navel orange Kinship is recent.It can be seen that there were similar functions during different members.DlDRM1 protein localized in the nucleus and cell membrane,whiles DlDRM2 protein localized in only nucleus.There were some differences in specific parameters-phosphorylation site,the coiled coil structure,and the protein secondary or tertiary structure between the two,and it was speculated that there may exist functional specificity.2 Subcellular localization analysis of longan DIDRMsBased on the sequences of DlDRM1 and DIDRM2 ORF,we consructed the 1302-DlDRM1-GFP and 1302-DlDRM2-GFP expression vectors.Fluorescence in onion epidermis was observed using fluorescence confocal microscopy post infection by Agrobacterium-mediated method.The results showed that DlDRM1 protein localized in the nucleus and cell membrane whereas DlDRM2 protein localized in only the nucleus,which is consistent with bioinformatics analysis.The nucleus is the critical site of DNA replication,which has important function on the cell cycle.The study of subcellular localization of DRM protein lays a foundation for further exploration of the function of this gene.3 Longan DIDRMs promoter verification and analysisThe 5'end sequence of the DlDRM1 and DIDRM2 genes extracted from the longan genome database was PCR-based,using the DNA of the embryogenic calli of longan as a template for cloning verification,and sequencesof 2594bp and 2354bp two sizes were obtained.The 5'-end sequences were identical to the genomic sequence.The alignment of sequences showed that DlDRM1 and DlDRM2 had low sequence homology,and their identity was 35.58%,indicating there may exist differences in their level of their evolution.To further understand the function of DlDRMs gene in longan,using the PlantCARE software to predict element their potential promoter of the 5' flanking sequence of the DlDRMs gene.The results showed that the DlDRMs gene promoter was rich in light,hormone,stress,and plant growthresponsive elements as well cis elements related toplant growth and development.It can be seen that the regulation of the expression of DIDRMs gene may be affected by many hormones and abiotic stress.4 qPCR analysis of longan DIDRMs under different tissue,development stages of somatic embryos,various exogenous hormones and abiotic stressThe DlDRMs gene were expressed in different tissues of longan(sijimi)and the expression trend was basically consistent.The highest expression level was in the pulp,followed by buds and then the leaves which recorded the lowest expression.In the same tissue,the expression of DlDRM1 gene was higher than that of the DlDRM2 gene.It can be seen that the expression of DlDRMs were not directly related to the maturity of tissues and organs.It can be speculated that DlDRMs are involved in the differentiation and formation of longan tissues and organs,and there are differences in DNA methylation level in different tissues of longan.The expression of DlDRM1 and DlDRM2 genes were not consistent in the development of longan somatic embryos.Among them,DlDRM1 was expressed in the non embryogenic and embryogenic culture of longan callus,and showed an overall downward trendl.The highest expression level was in non embryogenic callus,and the lowest in the cotyledonary embryo stage.the DlDRM2 gene at the transcriptional level was similar to the inverted "V"mode,the maximum amount of relative expression at ICpEC.It can be inferred that the non-embryonic and embryogenic transformation of longan callus may related to the expression of DlDRM1 gene,while the regulation of DlDRM2 was most significant in incomplete embryo compaction structure.DlDRM1 and DlDRM2 analysis upon treatment of longan embryogenic calli with 2,4-D,KT,SA,5-azac,GA3,and ABA hormones showed similar expression levels,but there were also some differences.With in a certain concentration range,exogenous hormones 2,4-D,SA and 5-azac can promote DlDRM1 expression,while KT,GA3 and ABA play an inhibitory role in DlDRM1 expression.Exogenous hormones 2,4-D,KT and SA can all enhanced the expression of DlDRM2,while 5-azac,GA3 and ABA significantly inhibited DlDRM2 expression.Under the abiotic stress treatments of sucrose,temperature,salt,and light quality,the expression trends of DlDRM1 and DlDRM2 were similar under the same conditions,and they may have similar functions.Under different illumination conditions,DIDRM2 was greatly affected by light,and the expression was decreased under the single illumination,and the expression of DlDRM1 was almost not affected by light change.Furthermore,the results of the experiment confirmed that the expression and regulation of DIDRMs gene play an important role in the growth and stress response of longan.5 Effect of DIDRMs gene silencing on the early stage of somatic embryogenesis in longanBased on the DlDRMs gene ORF sequences,siRNAs that knocked out the DIDRMs gene were designed and synthesized,followed by treatment of embryogenic calli in longan.qPCR assay results showed that DlDRM1-siRNA-314 and DlDRM2-siRNA-1599 had the best inhibitory effect.The selected siRNAs were used for the subsequent somatic embryo transformation experiments.The results showed that when DlDRM1 was only inhibited,the expression of DlDRM1 gene was down-regulated,but the expression of DlDRM2 gene showed a clear upward trend,and the developmental ability of longan somatic embryos did not change significantly.When DIDRM2 was only inhibited,the expression of DlDRM2 gene was down-regulated and the DlDRM1 gene expression was also down-regulated,but the down-regulation trend was no obvious change when DlDRM1 was inhibited alone,and the somatic embryogenesis rate of longan was slightly accelerated.At the same time,when DlDRM1 and DlDRM2 were inhibited,the expression of DlDRM1 and DlDRM2 were both decreased.The trends were all down-regulated,and the trend of DlDRMl's down-regulation was more significant than that of DlDRM1 alone,while the down-regulation of DlDRM2 was the same as the down-regulation of DIDRM2 alone,and the somatic embryo differentiation rate of longan was significantly faster.It is speculated that DlDRM2 plays a role in the regulation of DlDRM1 expression during somatic embryogenesis.In DlDRMs family,DIDRM2 can play a part of the function instead of DlDRM1.