Conserved miRNA analysis in Gossypium hirsutum through small RNA sequencing.

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.

Ruan MB ，Zhao YT ，Meng ZH ，Wang XJ ，Yang WC ... -  《-》

Identification of cotton microRNAs and their targets.

No study has been performed on identifying microRNAs (miRNAs) and their targets in cotton although cotton is one of the most important fiber and economic crops around the world. In this study, we found 30 potential cotton miRNAs using a comparative genomic approach based on genomic survey sequence analysis and miRNA secondary structure. These cotton miRNAs belong to 22 miRNA families. Expressed sequence tag (EST) analysis indicated that the predicted miRNAs were expressed in cotton plants. Based on the characteristic that miRNAs exhibit perfect or nearly perfect complementarity with their targeted mRNA sequences, a total of 139 potential miRNA targets were identified in cotton genome. A majority of these targets belong to transcriptional factors which regulate cotton growth and development, including leaf, root, stem, flower, and even fiber development. Those miRNAs may also be involved in other cellular and metabolic processes, such as stress response, signal transduction, and secondary wall synthesis and deposition. Some of the newly identified miRNA targets may be unique to cotton species. In this study, we found that at least 3 miRNA families (miR 396, 414, and 782) target callous synthase, fiber protein Fb23, and fiber quinone-oxidoreductase, suggesting that miRNAs play an important role in cotton fiber differentiation and development.

Zhang B ，Wang Q ，Wang K ，Pan X ，Liu F ，Guo T ，Cobb GP ，Anderson TA ... -  《GENE》

Computational identification of microRNAs and their targets in Gossypium hirsutum expressed sequence tags.

MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate gene post-transcriptional expression in animals and plants. Comparatively genomic computational methods have been developed to predict new miRNAs in worms, humans, and Arabidopsis. Here we present an EST (Expressed Sequence Tags)--and GSS (Genomic Survey Sequences)-based combined approach for the detection of novel miRNAs in Gossypium hirsutum. This was initiated by using previously known miRNA sequences from Arabidopsis, rice and other plant species and an algorithm called miRNAassist to blast the databases of G. hirsutum EST and GSS. A total of 37 potential miRNAs were detected following a range of filtering criteria. Using these potential miRNAs sequences, we further blasted the publicly available mRNA database and detected 96 potential targets in G. hirsutum. According to the mRNA information provided by the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), most of the miRNA targeted genes were predicted to encode transcription factors that regulate cell growth and development, signaling, and metabolism. So far, little is known about experimental or computational identification of miRNA in G. hirsutum species. These new miRNAs and their targets in G. hirsutum have been run through miRNAassist to yield data that may help us better understanding of the possible role of miRNAs in regulating the growth and development of G. hirsutum.

Qiu CX ，Xie FL ，Zhu YY ，Guo K ，Huang SQ ，Nie L ，Yang ZM ... -  《GENE》

Identification and characterization of new plant microRNAs using EST analysis.

Seventy-five previously known plant microRNAs (miRNAs) were classified into 14 families according to their gene sequence identity. A total of 18,694 plant expressed sequence tags (EST) were found in the GenBank EST databases by comparing all previously known Arabidopsis miRNAs to Genbank plant EST databases with BLAST algorithms. After removing the EST sequences with high numbers (more than 2) of mismatched nucleotides, a total of 812 EST contigs were identified. After predicting and scoring the RNA secondary structure of the 812 EST sequences using mFold software, 338 new potential miRNAs were identified in 60 plant species. miRNAs are widespread. Some microRNAs may highly conserve in the plant kingdom, and they may have the same ancestor in very early evolution. There is no nucleotide substitution in most miRNAs among many plant species. Some of the new identified potential miRNAs may be induced and regulated by environmental biotic and abiotic stresses. Some may be preferentially expressed in specific tissues, and are regulated by developmental switching. These findings suggest that EST analysis is a good alternative strategy for identifying new miRNA candidates, their targets, and other genes. A large number of miRNAs exist in different plant species and play important roles in plant developmental switching and plant responses to environmental abiotic and biotic stresses as well as signal transduction. Environmental stresses and developmental switching may be the signals for synthesis and regulation of miRNAs in plants. A model for miRNA induction and expression, and gene regulation by miRNA is hypothesized.

Zhang BH ，Pan XP ，Wang QL ，Cobb GP ，Anderson TA ... -  《-》

Identification of Taxus microRNAs and their targets with high-throughput sequencing and degradome analysis.

Plant microRNAs (miRNAs) have an impact in the regulation of several biological processes such as development, growth and metabolism by negatively controlling gene expression at the post-transcriptional level. However, the role of these small molecules in the medicinal gymnosperm species Taxus remained elusive. To elucidate the role of miRNAs in Taxus we used a deep sequencing approach to analyze the small RNA and degradome sequence tags of Taxus mairei leaves. For miRNAs, the sequencing library generated 14.9 million short sequences, resulting in 13.1 million clean reads. The library contains predominantly small RNAs with 21 nucleotide length, followed by 19-nt and 20-nt small RNAs. Around 29% of total small RNAs are matched to the T. mairei transcriptome. By sequence alignment, we identified 871 mature miRNAs, 15 miRNA* and 869 miRNA precursors representing known plant miRNA families. There are 547 unique small RNA matching the miRNA precursors. We predict 37 candidate novel miRNAs from the unannotated small RNAs that could be mapped to the reference transcriptome. The expression of the selected candidates was for the first time quantified by real-time reverse transcription polymerase chain reaction. The novel miRNA m0034 turns out to be from the intron sequence of the paclitaxel biosynthetic gene taxadiene synthase. The 21 potential targets of nine novel miRNAs are also predicted. Additionally, 56 targets for known miRNA families and 15 targets for novel candidate miRNA families were identified by high-throughput degradome-sequencing approach. It is found that two paclitaxel biosynthetic genes, taxane 13α hydroxylase and taxane 2α-O-benzoyltransferase, are the cleavage targets of miR164 and miR171, respectively. This study represents the first transcriptome-based analysis of miRNAs and degradome in gymnosperms.

Hao DC ，Yang L ，Xiao PG ，Liu M ... -  《-》