Comparative expression of miRNA genes and miRNA-based AFLP marker analysis in cultivated tetraploid cottons.
ABSTRACT MicroRNAs (miRNAs) are a class of small non-coding RNAs that down-regulate gene expression in a sequence specific manner to control plant growth and development. The identification and characterization of miRNAs are critical steps in finding their target genes and elucidating their functions. The objective of the present study was to assess the genetic variation of miRNA genes through expression comparisons and miRNA-based AFLP marker analysis. Seven miRNAs were first selected for RT-PCR and four for quantitative RT-PCR analysis that showed considerably high or differential expression levels in early stages of boll development. Except for miR160a, differential gene expression of miR171, 390a, and 396a was detected in early developing bolls at one or more timepoints between two cultivated cotton cultivars, Pima Phy 76 (Gossypium barbadense) and Acala 1517-99 (Gossypium hirsutum). Our further work demonstrated that genetic diversity of miRNA genes can be assessed by miRNA-AFLP analysis using primers designed from 22 conserved miRNA genes in combination with AFLP primers. Homologous miRNA genes can be also identified and isolated for sequencing and confirmation using this homology-based genotyping approach. This strategy offers an alternative to isolating a full length of miRNA genes and their up-stream and down-stream sequences. The significance of the expression and sequence differences of miRNAs between cotton species or genotypes needs further studies.
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ABSTRACT: MicroRNAs (miRNAs) are approximately 22-nt small non-coding RNAs that regulate the expression of specific target genes in many eukaryotes. In higher plants, miRNAs are involved in developmental processes and stress responses. Sexual reproduction in flowering plants relies on pollen, the male gametophyte, to deliver sperm cells to fertilize the egg cell hidden in the embryo sac. Studies indicated that post-transcriptional processes are important for regulating gene expression during pollen function. However, we still have very limited knowledge on the involved gene regulatory mechanisms. Especially, the function of miRNAs in pollen remains unknown. Using miRCURY LNA array technology, we have profiled the expression of 70 known miRNAs (representing 121 miRBase IDs) in Arabidopsis mature pollen, and compared the expression of these miRNAs in pollen and young inflorescence. Thirty-seven probes on the array were identified using RNAs isolated from mature pollen, 26 of which showed significant differences in expression between mature pollen and inflorescence. Real-time PCR based on TaqMan miRNA assays confirmed the expression of 22 miRNAs in mature pollen, and identified 8 additional miRNAs that were expressed at low level in mature pollen. However, the expression of 11 miRNA that were identified on the array could not be confirmed by the Taqman miRNA assays. Analyses of transcriptome data for some miRNA target genes indicated that miRNAs are functional in pollen. In summary, our results showed that some known miRNAs were expressed in Arabidopsis mature pollen, with most of them being low abundant. The results can be utilized in future research to study post-transcriptional gene regulation in pollen function.BMC Plant Biology 02/2009; 9:87. · 4.35 Impact Factor
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ABSTRACT: Several miRNA family and their targets in cotton had been identified by computational methods based on the conserved characterization of miRNAs. So far, there are no experiments to validate the existence of miRNAs in cotton. In this study, to analyze the miRNAs in cotton, a small RNA library of sequences from 18 to 26 nt of Gossypium hirsutum seedling has been built by high-throughput sequencing. In this library, 34 conserved miRNA families were identified by homology search and the miRNA sequences of them were also found in the library. Furthermore, potential targets of these conserved miRNA families were predicted in cotton TC library. However, based on the mature miRNAs and their miR sequences, only 8 conserved miRNA encoding loci (miR156, miR157a, miR157b, miR162, miR164, miR393, miR399, miR827) were identified from cotton EST sequences. Multiple encoding loci of some miRNAs were identified by comparing the cloned miRNA and miR sequences.Genomics 08/2009; 94(4):263-8. · 3.01 Impact Factor
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ABSTRACT: Background: Cotton fiber development undergoes rapid and dynamic changes in a single cell type, from fiber initiation, elongation, primary and secondary wall biosynthesis, to fiber maturation. Previous studies showed that cotton genes encoding putative MYB transcription factors and phytohormone responsive factors were induced during early stages of ovule and fiber development. Many of these factors are targets of microRNAs (miRNAs) that mediate target gene regulation by mRNA degradation or translational repression. Results: Here we sequenced and analyzed over 4 million small RNAs derived from fiber and nonfiber tissues in cotton. The 24-nucleotide small interfering RNAs (siRNAs) were more abundant and highly enriched in ovules and fiber-bearing ovules relative to leaves. A total of 31 miRNA families, including 27 conserved, 4 novel miRNA families and a candidate-novel miRNA, were identified in at least one of the cotton tissues examined. Among 32 miRNA precursors representing 19 unique miRNA families identified, 7 were previously reported, and 25 new miRNA precursors were found in this study. Sequencing, miRNA microarray, and small RNA blot analyses showed a trend of repression of miRNAs, including novel miRNAs, during ovule and fiber development, which correlated with upregulation of several target genes tested. Moreover, 223 targets of cotton miRNAs were predicted from the expressed sequence tags derived from cotton tissues, including ovules and fibers. The cotton miRNAs examined triggered cleavage in the predicted sites of the putative cotton targets in ovules and fibers. Conclusions: Enrichment of siRNAs in ovules and fibers suggests active small RNA metabolism and chromatin modifications during fiber development, whereas general repression of miRNAs in fibers correlates with upregulation of a dozen validated miRNA targets encoding transcription and phytohormone response factors, including the genes found to be highly expressed in cotton fibers. Rapid and dynamic changes in siRNAs and miRNAs may contribute to ovule and fiber development in allotetraploid cotton.Genome biology 11/2009; · 10.30 Impact Factor