Abstract
Elucidation of the molecular mechanism related to the dedifferentiation and redifferentiation during tissue culture will be useful for optimizing regeneration system of tea plant. In this study, an integrated sRNAome and transcriptome analyses were carried out during phase changes of the stem explant culture. Among 198 miRNAs and 8001 predicted target genes, 178 differentially expressed miRNAs and 4264 potential targets were screened out from explants, primary calli, as well as regenerated roots and shoots. According to KEGG analysis of the potential targets, pathway of “aminoacyl-tRNA biosynthesis”, “proteasome” and “glutathione metabolism” was of great significance during the dedifferentiation, and pathway of “porphyrin and chlorophyll metabolism”, “mRNA surveillance pathway”, “nucleotide excision repair” was indispensable for redifferentiation of the calli. Expression pattern of 12 miRNAs, including csn-micR390e, csn-miR156b-5p, csn-miR157d-5p, csn-miR156, csn-miR166a-3p, csn-miR166e, csn-miR167d, csn-miR393c-3p, csn-miR394, csn-miR396a-3p, csn-miR396 and csn-miR396e-3p, was validated by qRT-PCR among 57 differentially expressed phase-specific miRNAs. Validation also confirmed that regulatory module of csn-miR167d/ERF3, csn-miR156/SPB1, csn-miR166a-3p/ATHB15, csn-miR396/AIP15A, csn-miR157d-5p/GST and csn-miR393c-3p/ATG18b might play important roles in regulating the phase changes during tissue culture of stem explants.
Highlights
Elucidation of the molecular mechanism related to the dedifferentiation and redifferentiation during tissue culture will be useful for optimizing regeneration system of tea plant
A GhmiR157a-GhSPL10 regulatory module was proved to be associated with initial cellular dedifferentiation and callus proliferation via hormonal and flavonoid pathways14. miR166 could regulate the expression of the several class-III HD-ZIP genes and play an important role in the lateral root development[15]; and miR396 could modulate the transition of root stem cells into transit-amplifying cells through interacting with GROWTH-REGULATING FACTORs (GRFs) in Arabidopsis thaliana[16]
After small RNA (sRNA) sequencing, more than 21 M raw reads and 15 M clean reads were obtained from each biological sample, around 1/10 of clean reads could mapped onto the reference transcriptome unigenes, and the mapped reads were 2.76 M and 2.62 M, 2.13 M and 1.15 M in stem explants, primary-calli, regenerated roots and shoots, respectively (Table 2)
Summary
Elucidation of the molecular mechanism related to the dedifferentiation and redifferentiation during tissue culture will be useful for optimizing regeneration system of tea plant. According to an increasing number of published reports, miRNA-mediated regulation usually plays a critical role in the development of embryos, roots and shoots via the regulating transcription factors and hormone-related genes. In zygotic embryogenesis of Arabidopsis, miR165 and miR166 could control the transcript abundance of the PHABULOSA and PHAVOLUTA (PHB and PHV) genes which were the positive regulators of LEAFY COTYLEDON2 (LEC2), while miR160 could negatively target the AUXIN RESPONSE FACTORS including ARF10, ARF16 and ARF179. Mapped reads 2,760,666 2,617,031 2,125,327 1,145,331 and that miR156 and miR160 could modulate the shoot regeneration[20,21], while miR171 could influence the shoot branching[22] In light of these evidences, it would be important to identify the miRNAs present in tea plants. Investigation on mechanism of the dedifferentiation and redifferentiation during tissue culture might be useful for optimizing efficient regeneration system of tea plant
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