| Tissue culture is one of the basic means for preservation and propagation of rare germplasm resources,and also an important foundation for gene function and transgenic research.Up to now,the regeneration system of tea plant is far from perfection,which is mainly caused by unclear regulation mechanism of dedifferentiation and redifferentiation.In order to elucidate the epigenetic regulation mechanism of tissue dedifferentiation and redifferentiation in tea plants,the differential expression of functional genes and microRNAs and the methylation of genomic DNA during the dedifferentiation and redifferentiation processes of stem and leaf explants were studied through transcriptome and sRNA sequencing,qPCR and MASP.The main results obtained are as follows:(1)The results of transcriptome analysis and qPCR validation showed that the regulation of’plant hormone signal transduction’,’zeatin biosynthesis’,’DNA replication’,’glutathione metabolism’,’photosynthesis’ and ’secondary metabolic pathways’,were closely related to the dedifferentiation and redifferentiation of tea plant tissues.Auxin/cytokinin-related genes such as ARR5,GH3.1,IAA18,IAA29,CYCD3-1,CDKB2-2,MYB15,ARF18 and ERF RAP2-12,played significant roles in regulating hormone signaling pathways.The difference between the status of specialized tissues(such as stem segments,leaves,roots and shoots)and non-specialized tissues(primarily-callus,callus)might be determined by the differential expression patterns of relevant genes.Callus formation might be closely related to chloroplast degradation and cell proliferation induced by ethylene and cytokinin/auxin metabolism;root redifferentiation might be associated with ion transportation,polar growth,plastid remodeling;protein processing,photosynthesis,secondary metabolism and oxidative stress buffering might be responsible for bud inducing.(2)The results of sRNA sequencing showed that 204 microRNAs were detected in stem explants,callus as well as redifferentiated roots and shoots,while more than two-fold change of the 177 microRNAs expression was observed among the different samples.qPCR confirmed that 12 microRNAs(such as csn-miRNA-167d)were closely related to the dedifferentiation and redifferentiation of tissue culture in tea plants.Association analysis of miRNAs and their targeted genes showed that csn-miR167d,csn-miR156,csn-miR166a-3p,csn-miR396,csn-miR157d-5p and csn-miR393c-3p would significantly affect the dedifferentiation and redifferentiation through regulating the expression of the relevant targeted gene ERF3,SPB1,ATHB 15,AIP 15A,GST and ATG18b respectively.(3)MSAP analysis showed that the DNA methylation level of stem and leaf explants was higher than 45%,and that the level gradually decreased during callus formation.Similar methylation levels were observed in the stem callus-derived(35%)and leaf callus-derived roots(34%).Thirty-seven readable sequences with 34-334bp in length were obtained by recovering the methylation polymorphic loci,and more than 30 of them were highly homologous to the nuclear genomes of tea pant,tomato and Ipomoea indica.The sequencing results after treating with sodium bisulfite showed that a large number of cytosine methylation sites were achieved in the methylation-specific bands besides the restriction endonuclease recognition site,and the methylation status of these sites changed dynamically during the dedifferentiation and redifferentiation of the tea plant.However,the relationship among the changes of methylation specific sites,the expression of related genes as well as dedifferentiation and redifferentiation phenotypes needs to be further studied. |
