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Identification and characterization of cold-responsive microRNAs in tea plant (Camellia sinensis) and their targets using high-throughput sequencing and degradome analysis.

Zhang Y, Zhu X, Chen X, Song C, Zou Z, Wang Y, Wang M, Fang W, Li X - BMC Plant Biol. (2014)

Bottom Line: In addition, 215 potential candidate miRNAs were found, among, which 98 candidates with star sequences were chosen as novel miRNAs.The results were also confirmed by quantitative real-time polymerase chain reaction.The RLM-5'RACE procedure was successfully used to map the cleavage sites in six target genes of C. sinensis.

View Article: PubMed Central - PubMed

ABSTRACT

Background: MicroRNAs (miRNAs) are approximately 19 ~ 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs have critical functions in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interaction with specific target mRNAs. Camellia sinensis is one of the most important commercial beverage crops in the world. However, miRNAs associated with cold stress tolerance in C. sinensis remains unexplored. The use of high-throughput sequencing can provide a much deeper understanding of miRNAs. To obtain more insight into the function of miRNAs in cold stress tolerance, Illumina sequencing of C. sinensis sRNA was conducted.

Result: Solexa sequencing technology was used for high-throughput sequencing of the small RNA library from the cold treatment of tea leaves. To align the sequencing data with known plant miRNAs, we characterized 106 conserved C. sinensis miRNAs. In addition, 215 potential candidate miRNAs were found, among, which 98 candidates with star sequences were chosen as novel miRNAs. Both congruously and differentially regulated miRNAs were obtained, and cultivar-specific miRNAs were identified by microarray-based hybridization in response to cold stress. The results were also confirmed by quantitative real-time polymerase chain reaction. To confirm the targets of miRNAs, two degradome libraries from two treatments were constructed. According to degradome sequencing, 455 and 591 genes were identified as cleavage targets of miRNAs from cold treatments and control libraries, respectively, and 283 targets were present in both libraries. Functional analysis of these miRNA targets indicated their involvement in important activities, such as development, regulation of transcription, and stress response.

Conclusions: We discovered 31 up-regulated miRNAs and 43 down-regulated miRNAs in 'Yingshuang', and 46 up-regulated miRNA and 45 down-regulated miRNAs in 'Baiye 1' in response to cold stress, respectively. A total of 763 related target genes were detected by degradome sequencing. The RLM-5'RACE procedure was successfully used to map the cleavage sites in six target genes of C. sinensis. These findings reveal important information about the regulatory mechanism of miRNAs in C. sinensis, and promote the understanding of miRNA functions during the cold response. The miRNA genotype-specific expression model might explain the distinct cold sensitivities between tea lines.

No MeSH data available.


Related in: MedlinePlus

Length distribution of small RNA sequences obtained in the tea plant libraries.
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Fig1: Length distribution of small RNA sequences obtained in the tea plant libraries.

Mentions: Tea plants were stored at 4°C and 28°C for 1, 4, 8, 12, 24 and 48 h, respectively. A small RNA library of tea leaves, which was generated from a mixture of total RNAs from each cold-treatment stage, was subjected to high-throughput sequencing by the Illumina platform. Raw sequences were first subjected to an Illumina Pipeline filter provided by the supplier (Solexa 0.3). A total of 9,700,042 raw reads, representing 3,145,122 distinct sequences, were obtained. Reads without small RNA sequences, ranging from 15 nt to 30 nt in length, were filtered (Figure 1). The majority of the RNA sequences ranged from 19 nt to 25 nt in size. The most abundant small RNAs in the library were 24 nt long. The distribution of 24 nt small RNAs was approximately 45.96% and 69.67% in the total and unique sequences, respectively, whereas the distribution of 21 nt small RNAs in the total and unique sequences was approximately 13.68% and 5.67%, respectively. A total of 1,319,524 clean reads were obtained from the tea plant, including Rfam, rRNA, tRNA, snoRNA, snRNA, miRNA, other ncRNA, and repeats (Table 1). These clean reads were obtained by removing adaptor/acceptor sequences, filtering low quality tags, and cleaning the contaminants formed by adaptor-adaptor ligations and shorts RNAs less than 15 nt.Figure 1


Identification and characterization of cold-responsive microRNAs in tea plant (Camellia sinensis) and their targets using high-throughput sequencing and degradome analysis.

Zhang Y, Zhu X, Chen X, Song C, Zou Z, Wang Y, Wang M, Fang W, Li X - BMC Plant Biol. (2014)

Length distribution of small RNA sequences obtained in the tea plant libraries.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4209041&req=5

Fig1: Length distribution of small RNA sequences obtained in the tea plant libraries.
Mentions: Tea plants were stored at 4°C and 28°C for 1, 4, 8, 12, 24 and 48 h, respectively. A small RNA library of tea leaves, which was generated from a mixture of total RNAs from each cold-treatment stage, was subjected to high-throughput sequencing by the Illumina platform. Raw sequences were first subjected to an Illumina Pipeline filter provided by the supplier (Solexa 0.3). A total of 9,700,042 raw reads, representing 3,145,122 distinct sequences, were obtained. Reads without small RNA sequences, ranging from 15 nt to 30 nt in length, were filtered (Figure 1). The majority of the RNA sequences ranged from 19 nt to 25 nt in size. The most abundant small RNAs in the library were 24 nt long. The distribution of 24 nt small RNAs was approximately 45.96% and 69.67% in the total and unique sequences, respectively, whereas the distribution of 21 nt small RNAs in the total and unique sequences was approximately 13.68% and 5.67%, respectively. A total of 1,319,524 clean reads were obtained from the tea plant, including Rfam, rRNA, tRNA, snoRNA, snRNA, miRNA, other ncRNA, and repeats (Table 1). These clean reads were obtained by removing adaptor/acceptor sequences, filtering low quality tags, and cleaning the contaminants formed by adaptor-adaptor ligations and shorts RNAs less than 15 nt.Figure 1

Bottom Line: In addition, 215 potential candidate miRNAs were found, among, which 98 candidates with star sequences were chosen as novel miRNAs.The results were also confirmed by quantitative real-time polymerase chain reaction.The RLM-5'RACE procedure was successfully used to map the cleavage sites in six target genes of C. sinensis.

View Article: PubMed Central - PubMed

ABSTRACT

Background: MicroRNAs (miRNAs) are approximately 19 ~ 21 nucleotide noncoding RNAs produced by Dicer-catalyzed excision from stem-loop precursors. Many plant miRNAs have critical functions in development, nutrient homeostasis, abiotic stress responses, and pathogen responses via interaction with specific target mRNAs. Camellia sinensis is one of the most important commercial beverage crops in the world. However, miRNAs associated with cold stress tolerance in C. sinensis remains unexplored. The use of high-throughput sequencing can provide a much deeper understanding of miRNAs. To obtain more insight into the function of miRNAs in cold stress tolerance, Illumina sequencing of C. sinensis sRNA was conducted.

Result: Solexa sequencing technology was used for high-throughput sequencing of the small RNA library from the cold treatment of tea leaves. To align the sequencing data with known plant miRNAs, we characterized 106 conserved C. sinensis miRNAs. In addition, 215 potential candidate miRNAs were found, among, which 98 candidates with star sequences were chosen as novel miRNAs. Both congruously and differentially regulated miRNAs were obtained, and cultivar-specific miRNAs were identified by microarray-based hybridization in response to cold stress. The results were also confirmed by quantitative real-time polymerase chain reaction. To confirm the targets of miRNAs, two degradome libraries from two treatments were constructed. According to degradome sequencing, 455 and 591 genes were identified as cleavage targets of miRNAs from cold treatments and control libraries, respectively, and 283 targets were present in both libraries. Functional analysis of these miRNA targets indicated their involvement in important activities, such as development, regulation of transcription, and stress response.

Conclusions: We discovered 31 up-regulated miRNAs and 43 down-regulated miRNAs in 'Yingshuang', and 46 up-regulated miRNA and 45 down-regulated miRNAs in 'Baiye 1' in response to cold stress, respectively. A total of 763 related target genes were detected by degradome sequencing. The RLM-5'RACE procedure was successfully used to map the cleavage sites in six target genes of C. sinensis. These findings reveal important information about the regulatory mechanism of miRNAs in C. sinensis, and promote the understanding of miRNA functions during the cold response. The miRNA genotype-specific expression model might explain the distinct cold sensitivities between tea lines.

No MeSH data available.


Related in: MedlinePlus