Identification Of Cotton Micrornas And Their Targets By High-throughput Sequencing

The developmental process of cotton fibers is complex, and a large number of genes involved in regulation of fiber development. Between the past and the present, the researchers have explored the developmental mechanism on different levels of genes, proteins and post-translational modification, among them, miRNAs (microRNAs) have been widely concerned by researchers in recent years. miRNA (microRNA) was a21～24nt non-protein-coding small RNA, which plays an important role in controlling plant growth and development by directing target mRNA cleavage or translational inhibition. Many experiments demonstrated that miRNAs are closely related to fiber development, therefore, recognition and identification of fiber development-related miRNAs will be better for understanding the molecular mechanisms of cotton fiber development.In this study, ovules of0DPA (Days post-anthesis),3DPA and fibers of10DPA, which were provided from two lines of an interspecific BILs (Backcross inbred lines), were used to library construction and sequencing. We obtained the candidate miRNAs associated with cotton fibers elongation by separated and identified of small RNA, and we also verified the accuracy of the expression levels for miRNAs and their target genes by fluorescence quantitative PCR. Detailed results of this experiment are as follows:1. D5genome sequences and other published sequences of cotton were used for reference sequences, and we identified a total of561miRNAs, of all the561miRNAs,254miRNAs belonging to40miRNA families had sequence conservation and homology to miRNAs of other plant species,77were candidate known miRNAs and232were novel miRNAs.2. A total of142miRNAs, with101left by eliminating duplicates, were differentially expressed between the two samples (BILs). Among them,6conserved miRNAs and30new miRNAs were differentially expressed between the0DPA ovules of two samples,17conserved miRNAs and41new miRNAs were differentially expressed between the3DPA ovules of two samples, and10conserved miRNAs and38new miRNAs were differentially expressed between the10DPA fibers of two samples. New miRNAs account for76.8%of the total differentially expressed miRNA numbers, indicating that the novel miRNAs might make great contribution to fiber development and the study of these novel miRNAs was of great significance to exploring the mechanism of cotton fiber development.3. The reads sequenced by transcriptome were used for predicting of miRNA targets. Among40known miRNA families,231putative target genes were obtained as candidate target genes of33miRNA families. Among75candidate known miRNAs,218putative target genes were obtained as candidate target genes of35miRNAs. Among232candidate new miRNAs,746putative target genes were obtained as candidate target genes of152miRNAs. Expression profile analysis of miRNAs and their target genes indicate that most miRNAs act as a negative regulator of their target genes, but few were positive regulation of their target genes. The regulation model between miRNAs and their target genes are also different during different stages of cotton ovule epidermal cells development.4. We compared all target gene sequences against the KEGG protein databases using BLASTX. Target genes were significant enriched in plant hormone pathways and RIG-I-like receptor signaling pathways, and the most abundant transcripts were in auxin and brassinosteroid metabolism pathway. The former researches also indicated that auxin and brassinosteroid promoted both the fiber initiation and the elongation. So, these miRNAs may have important reference significance to explore the initiation and elongation mechanism on cotton fiber development.5.7miRNAs and its7target genes were validated by real-time fluorescence quantitative PCR, and they were clustered into four expression types. Among0DPA,3DPA ovules and10DPA fibers, miR164, miR168and miR172were up-regulated gradually; miR172was down-regulated gradually; miR390and miR4were up-regulated in NMGA-062, and they was down-regulated from0to3DPA and increased from3to10DPA in NMGA-105; miR211was up-regulated gradually in NMGA-062, and it was up-regulated from0to3DPA and reduced from3to10DPA in NMGA-105. The expression patterns of target genes were contrary with its miRNAs, except for miR172. The PCR results were coincident with sequence results, so, Transcriptome and miRNAome can effectively detect the expression levels of target genes and miRNAs, respectively. Combined Transcriptome and miRNAome can also intuitively reflect the regulation pattern between miRNAs and their targets, and they can provide an important basis for accurate prediction of miRNA target genes.