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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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Validation and expression of selected miRNA target genes.(A) 5′-RLM-RACE analysis of the cleavage of target mRNAs by corresponding miRNAs. The arrows indicate the cleavage sites, and the numbers represent the frequency of the sequenced clones. (B) Expression profiling analysis of several target genes and their corresponding miRNAs in rice grain on different days after fertilization. Actin was used as a reference, and the expression levels of each of the target mRNAs were then compared with their expression at 5 DAF or 12 DAF, which was set to 1.0. Error bars indicate the standard deviation (±SD) of three replicates. Os04g47870 (PINHEAD) and Os12g41680 (no apical meristem protein) were confirmed to be targets of Osa-miR168 and Osa-miR164, respectively, in previous work [59]. Os01g63290 (transporter) was predicted to be target of osa-miR167d/f-h/j using psRNA Target (data not shown).
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pone-0057863-g004: Validation and expression of selected miRNA target genes.(A) 5′-RLM-RACE analysis of the cleavage of target mRNAs by corresponding miRNAs. The arrows indicate the cleavage sites, and the numbers represent the frequency of the sequenced clones. (B) Expression profiling analysis of several target genes and their corresponding miRNAs in rice grain on different days after fertilization. Actin was used as a reference, and the expression levels of each of the target mRNAs were then compared with their expression at 5 DAF or 12 DAF, which was set to 1.0. Error bars indicate the standard deviation (±SD) of three replicates. Os04g47870 (PINHEAD) and Os12g41680 (no apical meristem protein) were confirmed to be targets of Osa-miR168 and Osa-miR164, respectively, in previous work [59]. Os01g63290 (transporter) was predicted to be target of osa-miR167d/f-h/j using psRNA Target (data not shown).

Mentions: Among the predicted targets of the miRNAs, cleavage events associated with three transcripts of the targets were chosen to be validated by 5′-RACE. Similar to other studies, the mRNAs of the SBP-box gene family member OsSPL14 (Os08g39890) and START domain-containing protein genes (Os03g01890 and Os03g43930) were cleaved within the complementary regions of osa-miR156a-j and osa-miR166a-d/f/n, respectively, and were precisely terminated at the 10th position relative to the 5′ end of the complementary regions of osa-miR156a-j and osa-miR166a-d/f/n (Fig. 4A).


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)

Validation and expression of selected miRNA target genes.(A) 5′-RLM-RACE analysis of the cleavage of target mRNAs by corresponding miRNAs. The arrows indicate the cleavage sites, and the numbers represent the frequency of the sequenced clones. (B) Expression profiling analysis of several target genes and their corresponding miRNAs in rice grain on different days after fertilization. Actin was used as a reference, and the expression levels of each of the target mRNAs were then compared with their expression at 5 DAF or 12 DAF, which was set to 1.0. Error bars indicate the standard deviation (±SD) of three replicates. Os04g47870 (PINHEAD) and Os12g41680 (no apical meristem protein) were confirmed to be targets of Osa-miR168 and Osa-miR164, respectively, in previous work [59]. Os01g63290 (transporter) was predicted to be target of osa-miR167d/f-h/j using psRNA Target (data not shown).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585941&req=5

pone-0057863-g004: Validation and expression of selected miRNA target genes.(A) 5′-RLM-RACE analysis of the cleavage of target mRNAs by corresponding miRNAs. The arrows indicate the cleavage sites, and the numbers represent the frequency of the sequenced clones. (B) Expression profiling analysis of several target genes and their corresponding miRNAs in rice grain on different days after fertilization. Actin was used as a reference, and the expression levels of each of the target mRNAs were then compared with their expression at 5 DAF or 12 DAF, which was set to 1.0. Error bars indicate the standard deviation (±SD) of three replicates. Os04g47870 (PINHEAD) and Os12g41680 (no apical meristem protein) were confirmed to be targets of Osa-miR168 and Osa-miR164, respectively, in previous work [59]. Os01g63290 (transporter) was predicted to be target of osa-miR167d/f-h/j using psRNA Target (data not shown).
Mentions: Among the predicted targets of the miRNAs, cleavage events associated with three transcripts of the targets were chosen to be validated by 5′-RACE. Similar to other studies, the mRNAs of the SBP-box gene family member OsSPL14 (Os08g39890) and START domain-containing protein genes (Os03g01890 and Os03g43930) were cleaved within the complementary regions of osa-miR156a-j and osa-miR166a-d/f/n, respectively, and were precisely terminated at the 10th position relative to the 5′ end of the complementary regions of osa-miR156a-j and osa-miR166a-d/f/n (Fig. 4A).

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