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Identification and characterization of miRNAs in ripening fruit of Lycium barbarum L. using high-throughput sequencing.

Zeng S, Liu Y, Pan L, Hayward A, Wang Y - Front Plant Sci (2015)

Bottom Line: Of the novel miRNAs, 36 were specific to L. barbarum fruits compared with L. chinense.These include miR156 targeting of fructokinase and 1-deoxy-D-xylulose-5-phosphate synthase and miR164 targeting of beta-fructofuranosidase.In sum, valuable information revealed by small RNA sequencing in this study will provide a solid foundation for uncovering the miRNA-mediated mechanism of fruit ripening and quality in this nutritional food.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences Guangzhou, China ; Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences Guangzhou, China.

ABSTRACT
MicroRNAs (miRNAs) are master regulators of gene activity documented to play central roles in fruit ripening in model plant species, yet little is known of their roles in Lycium barbarum L. fruits. In this study, miRNA levels in L. barbarum fruit samples at four developmental stages, were assayed using Illumina HiSeqTM2000. This revealed the presence of 50 novel miRNAs and 38 known miRNAs in L. barbarum fruits. Of the novel miRNAs, 36 were specific to L. barbarum fruits compared with L. chinense. A number of stage-specific miRNAs were identified and GO terms were assigned to 194 unigenes targeted by miRNAs. The majority of GO terms of unigenes targeted by differentially expressed miRNAs are "intracellular organelle," "binding," "metabolic process," "pigmentation," and "biological regulation." Enriched KEGG analysis indicated that nucleotide excision repair and ubiquitin mediated proteolysis were over-represented during the initial stage of ripening, with ABC transporters and sulfur metabolism pathways active during the middle stages and ABC transporters and spliceosome enriched in the final stages of ripening. Several miRNAs and their targets serving as potential regulators in L. barbarum fruit ripening were identified using quantitative reverse transcription polymerase chain reaction. The miRNA-target interactions were predicted for L. barbarum ripening regulators including miR156/157 with LbCNR and LbWRKY8, and miR171 with LbGRAS. Additionally, regulatory interactions potentially controlling fruit quality and nutritional value via sugar and secondary metabolite accumulation were identified. These include miR156 targeting of fructokinase and 1-deoxy-D-xylulose-5-phosphate synthase and miR164 targeting of beta-fructofuranosidase. In sum, valuable information revealed by small RNA sequencing in this study will provide a solid foundation for uncovering the miRNA-mediated mechanism of fruit ripening and quality in this nutritional food.

No MeSH data available.


Related in: MedlinePlus

Expression profile of miRNAs and their target genes in ripening fruits of Lycium barbarum L. The total RNAs were isolated from fruits at four developmental stages referring to S1 stage (green fruit, 3 days before color break), S2 stage (the color-break stage), S3 stage (light-color, 3 days after color break), and S4 stages (ripe fruit, 6 days after color break). These results were presented as mean ± SD of three biological replicates. The LbβFFase is predicted to be the target gene of miR164d, as well as LbGRAS for miR171a, Lbcid14 for miR5538, and LbOACPT for miR167a. The expression profile of miR164d, miR171a, miR167a, and miR5538 tested by qRT-PCR was identical to that of small RNA sequencing.
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Figure 6: Expression profile of miRNAs and their target genes in ripening fruits of Lycium barbarum L. The total RNAs were isolated from fruits at four developmental stages referring to S1 stage (green fruit, 3 days before color break), S2 stage (the color-break stage), S3 stage (light-color, 3 days after color break), and S4 stages (ripe fruit, 6 days after color break). These results were presented as mean ± SD of three biological replicates. The LbβFFase is predicted to be the target gene of miR164d, as well as LbGRAS for miR171a, Lbcid14 for miR5538, and LbOACPT for miR167a. The expression profile of miR164d, miR171a, miR167a, and miR5538 tested by qRT-PCR was identical to that of small RNA sequencing.

Mentions: To validate the expression profiles of miRNAs in ripening fruits of L. barbarum, 13 significant miRNAs were investigated using stem-loop qRT-PCR (Figures 4, 6, 7 and Figure S10). Among these, the expression profiles of miR164d, miR171a, miR167a, miR403, miR5538, miR09, and miR40 were consistent with the results of small RNA sequencing (Figures 4, 6, 7). As shown in Figures 6, 7, miR164d, miR171a, miR167a, and miR156 transcripts were expressed highly in S2 and decreased subsequently. In contrast, miR5538 transcripts decreased from S1 to S2, then increased to the highest level at S3 followed by decrease at S4.


Identification and characterization of miRNAs in ripening fruit of Lycium barbarum L. using high-throughput sequencing.

Zeng S, Liu Y, Pan L, Hayward A, Wang Y - Front Plant Sci (2015)

Expression profile of miRNAs and their target genes in ripening fruits of Lycium barbarum L. The total RNAs were isolated from fruits at four developmental stages referring to S1 stage (green fruit, 3 days before color break), S2 stage (the color-break stage), S3 stage (light-color, 3 days after color break), and S4 stages (ripe fruit, 6 days after color break). These results were presented as mean ± SD of three biological replicates. The LbβFFase is predicted to be the target gene of miR164d, as well as LbGRAS for miR171a, Lbcid14 for miR5538, and LbOACPT for miR167a. The expression profile of miR164d, miR171a, miR167a, and miR5538 tested by qRT-PCR was identical to that of small RNA sequencing.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585183&req=5

Figure 6: Expression profile of miRNAs and their target genes in ripening fruits of Lycium barbarum L. The total RNAs were isolated from fruits at four developmental stages referring to S1 stage (green fruit, 3 days before color break), S2 stage (the color-break stage), S3 stage (light-color, 3 days after color break), and S4 stages (ripe fruit, 6 days after color break). These results were presented as mean ± SD of three biological replicates. The LbβFFase is predicted to be the target gene of miR164d, as well as LbGRAS for miR171a, Lbcid14 for miR5538, and LbOACPT for miR167a. The expression profile of miR164d, miR171a, miR167a, and miR5538 tested by qRT-PCR was identical to that of small RNA sequencing.
Mentions: To validate the expression profiles of miRNAs in ripening fruits of L. barbarum, 13 significant miRNAs were investigated using stem-loop qRT-PCR (Figures 4, 6, 7 and Figure S10). Among these, the expression profiles of miR164d, miR171a, miR167a, miR403, miR5538, miR09, and miR40 were consistent with the results of small RNA sequencing (Figures 4, 6, 7). As shown in Figures 6, 7, miR164d, miR171a, miR167a, and miR156 transcripts were expressed highly in S2 and decreased subsequently. In contrast, miR5538 transcripts decreased from S1 to S2, then increased to the highest level at S3 followed by decrease at S4.

Bottom Line: Of the novel miRNAs, 36 were specific to L. barbarum fruits compared with L. chinense.These include miR156 targeting of fructokinase and 1-deoxy-D-xylulose-5-phosphate synthase and miR164 targeting of beta-fructofuranosidase.In sum, valuable information revealed by small RNA sequencing in this study will provide a solid foundation for uncovering the miRNA-mediated mechanism of fruit ripening and quality in this nutritional food.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences Guangzhou, China ; Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences Guangzhou, China.

ABSTRACT
MicroRNAs (miRNAs) are master regulators of gene activity documented to play central roles in fruit ripening in model plant species, yet little is known of their roles in Lycium barbarum L. fruits. In this study, miRNA levels in L. barbarum fruit samples at four developmental stages, were assayed using Illumina HiSeqTM2000. This revealed the presence of 50 novel miRNAs and 38 known miRNAs in L. barbarum fruits. Of the novel miRNAs, 36 were specific to L. barbarum fruits compared with L. chinense. A number of stage-specific miRNAs were identified and GO terms were assigned to 194 unigenes targeted by miRNAs. The majority of GO terms of unigenes targeted by differentially expressed miRNAs are "intracellular organelle," "binding," "metabolic process," "pigmentation," and "biological regulation." Enriched KEGG analysis indicated that nucleotide excision repair and ubiquitin mediated proteolysis were over-represented during the initial stage of ripening, with ABC transporters and sulfur metabolism pathways active during the middle stages and ABC transporters and spliceosome enriched in the final stages of ripening. Several miRNAs and their targets serving as potential regulators in L. barbarum fruit ripening were identified using quantitative reverse transcription polymerase chain reaction. The miRNA-target interactions were predicted for L. barbarum ripening regulators including miR156/157 with LbCNR and LbWRKY8, and miR171 with LbGRAS. Additionally, regulatory interactions potentially controlling fruit quality and nutritional value via sugar and secondary metabolite accumulation were identified. These include miR156 targeting of fructokinase and 1-deoxy-D-xylulose-5-phosphate synthase and miR164 targeting of beta-fructofuranosidase. In sum, valuable information revealed by small RNA sequencing in this study will provide a solid foundation for uncovering the miRNA-mediated mechanism of fruit ripening and quality in this nutritional food.

No MeSH data available.


Related in: MedlinePlus