MicroRNAs and their targeted genes associated with phase changes of stem explants during tissue culture of tea plant

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.

Figure 1

Venn diagram of sRNA common and unique sequence number among various samples. The types of unique sequences (represented by Unique reads) and the number of common sequences (represented by Total reads) between different samples were counted. The sRNA sequence types were counted with the reads after removal of redundancy; the number of sRNA sequences was counted by all reads. (A) S_Explant (S01) vs S_Primary callus (S02); (B) S_Primary callus (S02) vs S_Root (S03); (C) S_Primary callus (S02) vs S_Shoot (S04).

Figure 2

The heat-map of 178 different expressed miRNA shared in the 4 samples, based on Z-score normalized TPM values in eight internode segments.

Figure 3

KEGG analysis of the targeted genes during phase change of tissue culture. (A) S_Explant vs S_Primary callus; (B) S_Root vs S_Primary callus; (C) S_Primary callus vs S_Shoot.

Figure 4

(A) expression validation of phase-specific miRNA by qPCR; (B) expression relationship between 6 phase-specific miRNAs and their targeted genes, the line represented the expression level of miRNAs and corresponded to the principal ordinate axis (left Y-axis), the histogram represented expression level of the targeted genes and corresponded to the secondary ordinate axis (right Y-axis). Different letters indicated significant differences at p < 0.05 (lower letters for miRNA expression and uppercase for targeted gene expression).

Figure 5

Potential regulatory module of phase-specific miRNA and the targeted genes during phase change of the tissue culture of stem explants.