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Identification and characterization of microRNAs from Chinese pollination constant non-astringent persimmon using high-throughput sequencing.

Luo Y, Zhang X, Luo Z, Zhang Q, Liu J - BMC Plant Biol. (2015)

Bottom Line: microRNAs (miRNAs) have been shown to play key roles in regulating gene expression at post-transcriptional level, but miRNAs associated with natural deastringency of Chinese pollination-constant nonastringent persimmon (CPCNA) have never been identified.In addition, dka-miR396g and miR2911a may regulate their target genes associated with glucosylation and insolubilization of tannin precursors.All of these miRNAs might play key roles in the regulation of (de)astringency in persimmon fruits under normal development conditions.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Horticultural Plant Biology (MOE), College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, 430070, China. luoyujie200701@163.com.

ABSTRACT

Background: microRNAs (miRNAs) have been shown to play key roles in regulating gene expression at post-transcriptional level, but miRNAs associated with natural deastringency of Chinese pollination-constant nonastringent persimmon (CPCNA) have never been identified.

Results: In this study, two small RNA libraries established using 'Eshi No. 1' persimmon (Diospyros kaki Thunb.; CPCNA) fruits collected at 15 and 20 weeks after flowering (WAF) were sequenced through Solexa platform in order to identify miRNAs involved in deastringency of persimmon. A total of 6,258,487 and 7,634,169 reads were generated for the libraries at 15 and 20 WAF, respectively. Based on sequence similarity and hairpin structure prediction, 236 known miRNAs belonging to 65 miRNA families and 33 novel miRNAs were identified using persimmon transcriptome data. Sixty one of the characterized miRNAs exhibited pronounced difference in the expression levels between 15 and 20 WAF, 17 up-regulated and 44 down-regulated. Expression profiles of 12 conserved and 10 novel miRNAs were validated by stem loop qRT-PCR. A total of 198 target genes were predicted for the differentially expressed miRNAs, including several genes that have been reported to be implicated in proanthocyanidins (PAs, or called tannin) accumulation. In addition, two transcription factors, a GRF and a bHLH, were experimentally confirmed as the targets of dka-miR396 and dka-miR395, respectively.

Conclusions: Taken together, the present data unraveled several important miRNAs in persimmon. Among them, miR395p-3p and miR858b may regulate bHLH and MYB, respectively, which are influenced by SPL under the control of miR156j-5p and in turn regulate the structural genes involved in PA biosynthesis. In addition, dka-miR396g and miR2911a may regulate their target genes associated with glucosylation and insolubilization of tannin precursors. All of these miRNAs might play key roles in the regulation of (de)astringency in persimmon fruits under normal development conditions.

No MeSH data available.


Validation of the differentially expressed miRNAs identified by deep sequencing. Expression analysis of known (A) and novel (B) miRNAs by stem loop qRT-PCR.
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Fig4: Validation of the differentially expressed miRNAs identified by deep sequencing. Expression analysis of known (A) and novel (B) miRNAs by stem loop qRT-PCR.

Mentions: Stem-loop qRT-PCR, which is a reliable method for assessing miRNA expression levels, has been applied for experimental verification of the miRNAs [27,33]. For this purpose, we analyzed expression of 22 miRNAs, including 12 randomly selected known miRNAs (dka-miR159e-5p, dka-miR396g, dka-miR2111d, dka-miR530b, dka-miR858b, dka-miR164d, dka-miR156a, dka-miR156j, dka-miR160a, dka-miR398a, dka-miR535c, and dka-miR827-3p) and 10 novel miRNAs with relatively high expression levels (miRN03, miRN07, miRN12, miRN15, miRN16, miRN23, miRN25, miRN28, miRN31, and miRN33. The qRT-PCR analysis showed that expression patterns of the examined miRNAs (FigureĀ 4A, B) were largely consistent with the results of deep sequencing except dka-miR156a, dka-miRN16, and dka-miRN28, which displayed opposite profiles between the two methods.Figure 4


Identification and characterization of microRNAs from Chinese pollination constant non-astringent persimmon using high-throughput sequencing.

Luo Y, Zhang X, Luo Z, Zhang Q, Liu J - BMC Plant Biol. (2015)

Validation of the differentially expressed miRNAs identified by deep sequencing. Expression analysis of known (A) and novel (B) miRNAs by stem loop qRT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4308916&req=5

Fig4: Validation of the differentially expressed miRNAs identified by deep sequencing. Expression analysis of known (A) and novel (B) miRNAs by stem loop qRT-PCR.
Mentions: Stem-loop qRT-PCR, which is a reliable method for assessing miRNA expression levels, has been applied for experimental verification of the miRNAs [27,33]. For this purpose, we analyzed expression of 22 miRNAs, including 12 randomly selected known miRNAs (dka-miR159e-5p, dka-miR396g, dka-miR2111d, dka-miR530b, dka-miR858b, dka-miR164d, dka-miR156a, dka-miR156j, dka-miR160a, dka-miR398a, dka-miR535c, and dka-miR827-3p) and 10 novel miRNAs with relatively high expression levels (miRN03, miRN07, miRN12, miRN15, miRN16, miRN23, miRN25, miRN28, miRN31, and miRN33. The qRT-PCR analysis showed that expression patterns of the examined miRNAs (FigureĀ 4A, B) were largely consistent with the results of deep sequencing except dka-miR156a, dka-miRN16, and dka-miRN28, which displayed opposite profiles between the two methods.Figure 4

Bottom Line: microRNAs (miRNAs) have been shown to play key roles in regulating gene expression at post-transcriptional level, but miRNAs associated with natural deastringency of Chinese pollination-constant nonastringent persimmon (CPCNA) have never been identified.In addition, dka-miR396g and miR2911a may regulate their target genes associated with glucosylation and insolubilization of tannin precursors.All of these miRNAs might play key roles in the regulation of (de)astringency in persimmon fruits under normal development conditions.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Horticultural Plant Biology (MOE), College of Horticulture and Forestry Science, Huazhong Agricultural University, Wuhan, 430070, China. luoyujie200701@163.com.

ABSTRACT

Background: microRNAs (miRNAs) have been shown to play key roles in regulating gene expression at post-transcriptional level, but miRNAs associated with natural deastringency of Chinese pollination-constant nonastringent persimmon (CPCNA) have never been identified.

Results: In this study, two small RNA libraries established using 'Eshi No. 1' persimmon (Diospyros kaki Thunb.; CPCNA) fruits collected at 15 and 20 weeks after flowering (WAF) were sequenced through Solexa platform in order to identify miRNAs involved in deastringency of persimmon. A total of 6,258,487 and 7,634,169 reads were generated for the libraries at 15 and 20 WAF, respectively. Based on sequence similarity and hairpin structure prediction, 236 known miRNAs belonging to 65 miRNA families and 33 novel miRNAs were identified using persimmon transcriptome data. Sixty one of the characterized miRNAs exhibited pronounced difference in the expression levels between 15 and 20 WAF, 17 up-regulated and 44 down-regulated. Expression profiles of 12 conserved and 10 novel miRNAs were validated by stem loop qRT-PCR. A total of 198 target genes were predicted for the differentially expressed miRNAs, including several genes that have been reported to be implicated in proanthocyanidins (PAs, or called tannin) accumulation. In addition, two transcription factors, a GRF and a bHLH, were experimentally confirmed as the targets of dka-miR396 and dka-miR395, respectively.

Conclusions: Taken together, the present data unraveled several important miRNAs in persimmon. Among them, miR395p-3p and miR858b may regulate bHLH and MYB, respectively, which are influenced by SPL under the control of miR156j-5p and in turn regulate the structural genes involved in PA biosynthesis. In addition, dka-miR396g and miR2911a may regulate their target genes associated with glucosylation and insolubilization of tannin precursors. All of these miRNAs might play key roles in the regulation of (de)astringency in persimmon fruits under normal development conditions.

No MeSH data available.