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Identification and expression analysis of microRNAs at the grain filling stage in rice(Oryza sativa L.)via deep sequencing.

Yi R, Zhu Z, Hu J, Qian Q, Dai J, Ding Y - PLoS ONE (2013)

Bottom Line: A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain.In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally.Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Hybrid Rice, Department of Genetics, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.

ABSTRACT
MicroRNAs (miRNAs) have been shown to play crucial roles in the regulation of plant development. In this study, high-throughput RNA-sequencing technology was used to identify novel miRNAs, and to reveal miRNAs expression patterns at different developmental stages during rice (Oryza sativa L.) grain filling. A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain. The expression profiles of these identified miRNAs were analyzed and the results showed that 161 known miRNAs were differentially expressed during grain development, a high proportion of which were up-regulated from 5 to 7 days after fertilization. In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally. Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation. In this study, differences were revealed in the composition and expression profiles of miRNAs among individual developmental stages during the rice grain filling process, and miRNA editing events were also observed, analyzed and validated during this process. The results provide novel insight into the dynamic profiles of miRNAs in developing rice grain and contribute to the understanding of the regulatory roles of miRNAs in grain filling.

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Predicted novel miRNAs identified in this study.(A) Predicted stem-loop structures of novel miRNA precursors. The precursor structures of four newly identified rice miRNAs (Osa-2, Osa-14, Osa-35 and Osa-46) were predicted via the MFOLD pipeline. Mature miRNA and miRNA* sequences are highlighted in red and blue, respectively. The numbers along the structure indicate nucleotide sites from the 5′ end of the pre-miRNAs sequence. (B) Stem-loop RT-PCR analysis if the identified novel miRNAs. Five novel miRNAs were confirmed via stem-loop RT-PCR. The sizes of the obtained PCR products were approximately ∼60 bp. M indicates a 20 bp DNA Ladder Marker (Takara, Japan). The arrow indicates 60 bp.
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pone-0057863-g002: Predicted novel miRNAs identified in this study.(A) Predicted stem-loop structures of novel miRNA precursors. The precursor structures of four newly identified rice miRNAs (Osa-2, Osa-14, Osa-35 and Osa-46) were predicted via the MFOLD pipeline. Mature miRNA and miRNA* sequences are highlighted in red and blue, respectively. The numbers along the structure indicate nucleotide sites from the 5′ end of the pre-miRNAs sequence. (B) Stem-loop RT-PCR analysis if the identified novel miRNAs. Five novel miRNAs were confirmed via stem-loop RT-PCR. The sizes of the obtained PCR products were approximately ∼60 bp. M indicates a 20 bp DNA Ladder Marker (Takara, Japan). The arrow indicates 60 bp.

Mentions: One of the most important advantages of high-throughput sequencing technology is that it can produce a large volume of data up to the gigabase level during small RNA sequencing, which is helpful for detecting novel miRNAs with extremely low expression levels. In this study, a total of 60 predicted novel miRNAs were obtained (Table S3), fifty-six of which have not been deposited in miRBase (v18.0) and have never been detected in Oryza sativa or Arabidopsis thaliana, while 4 of these miRNAs were reported during preparation of this draft [20], [23], [31]. The structures of the precursors of all of the novel miRNAs were predicted using MFOLD (http://mfold.rna.albany.edu/) and checked manually. Four of these structures are presented (Fig. 2A). The novel miRNAs were temporarily named following the Osa-number format, e.g., Osa-1, before being submitted to obtain an official designation.


Identification and expression analysis of microRNAs at the grain filling stage in rice(Oryza sativa L.)via deep sequencing.

Yi R, Zhu Z, Hu J, Qian Q, Dai J, Ding Y - PLoS ONE (2013)

Predicted novel miRNAs identified in this study.(A) Predicted stem-loop structures of novel miRNA precursors. The precursor structures of four newly identified rice miRNAs (Osa-2, Osa-14, Osa-35 and Osa-46) were predicted via the MFOLD pipeline. Mature miRNA and miRNA* sequences are highlighted in red and blue, respectively. The numbers along the structure indicate nucleotide sites from the 5′ end of the pre-miRNAs sequence. (B) Stem-loop RT-PCR analysis if the identified novel miRNAs. Five novel miRNAs were confirmed via stem-loop RT-PCR. The sizes of the obtained PCR products were approximately ∼60 bp. M indicates a 20 bp DNA Ladder Marker (Takara, Japan). The arrow indicates 60 bp.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585941&req=5

pone-0057863-g002: Predicted novel miRNAs identified in this study.(A) Predicted stem-loop structures of novel miRNA precursors. The precursor structures of four newly identified rice miRNAs (Osa-2, Osa-14, Osa-35 and Osa-46) were predicted via the MFOLD pipeline. Mature miRNA and miRNA* sequences are highlighted in red and blue, respectively. The numbers along the structure indicate nucleotide sites from the 5′ end of the pre-miRNAs sequence. (B) Stem-loop RT-PCR analysis if the identified novel miRNAs. Five novel miRNAs were confirmed via stem-loop RT-PCR. The sizes of the obtained PCR products were approximately ∼60 bp. M indicates a 20 bp DNA Ladder Marker (Takara, Japan). The arrow indicates 60 bp.
Mentions: One of the most important advantages of high-throughput sequencing technology is that it can produce a large volume of data up to the gigabase level during small RNA sequencing, which is helpful for detecting novel miRNAs with extremely low expression levels. In this study, a total of 60 predicted novel miRNAs were obtained (Table S3), fifty-six of which have not been deposited in miRBase (v18.0) and have never been detected in Oryza sativa or Arabidopsis thaliana, while 4 of these miRNAs were reported during preparation of this draft [20], [23], [31]. The structures of the precursors of all of the novel miRNAs were predicted using MFOLD (http://mfold.rna.albany.edu/) and checked manually. Four of these structures are presented (Fig. 2A). The novel miRNAs were temporarily named following the Osa-number format, e.g., Osa-1, before being submitted to obtain an official designation.

Bottom Line: A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain.In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally.Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Hybrid Rice, Department of Genetics, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China.

ABSTRACT
MicroRNAs (miRNAs) have been shown to play crucial roles in the regulation of plant development. In this study, high-throughput RNA-sequencing technology was used to identify novel miRNAs, and to reveal miRNAs expression patterns at different developmental stages during rice (Oryza sativa L.) grain filling. A total of 434 known miRNAs (380, 402, 390 and 392 at 5, 7, 12 and 17 days after fertilization, respectively.) were obtained from rice grain. The expression profiles of these identified miRNAs were analyzed and the results showed that 161 known miRNAs were differentially expressed during grain development, a high proportion of which were up-regulated from 5 to 7 days after fertilization. In addition, sixty novel miRNAs were identified, and five of these were further validated experimentally. Additional analysis showed that the predicted targets of the differentially expressed miRNAs may participate in signal transduction, carbohydrate and nitrogen metabolism, the response to stimuli and epigenetic regulation. In this study, differences were revealed in the composition and expression profiles of miRNAs among individual developmental stages during the rice grain filling process, and miRNA editing events were also observed, analyzed and validated during this process. The results provide novel insight into the dynamic profiles of miRNAs in developing rice grain and contribute to the understanding of the regulatory roles of miRNAs in grain filling.

Show MeSH