Jin-Yuan Liu

A Comparative miRNAome Analysis Reveals Seven Fiber Initiation-Related and 36 Novel miRNAs in Developing Cotton Ovules

An increasing number of microRNAs (miRNAs) have been shown to play crucial regulatory roles in the process of plant development. Here, we used high-throughput sequencing combined with computational analysis to characterize miRNAomes from the ovules of wild-type upland cotton and a fiberless mutant during fiber initiation. Comparative miRNAome analysis combined with northern blotting and RACE-PCR revealed seven fiber initiation-related miRNAs expressed in cotton ovules and experimentally validated targets of these miRNAs are involved in different cellular responses and metabolic processes, including transcriptional regulation, auxin and gibberellin signal transduction, actin bundles, and lignin biosynthesis. This paper describes a complex regulatory network consisting of these miRNAs expressed in cotton ovules to coordinate fiber initiation responses. In addition, 36 novel miRNAs and two conserved miRNAs were newly identified, nearly doubling the number of known cotton miRNA families to a total of 78. Furthermore, a chromatin remodeling complex subunit and a pre-mRNA splicing factor are shown for the first time to be miRNA targets. To our knowledge, this study is the first systematic investigation of fiber initiation-related miRNAs and their targets in the developing cotton ovule, deepening our understanding of the important regulatory functions of miRNAs in cotton fiber initiation.

Genome-wide analysis of small RNAs reveals eight fiber elongation-related and 257 novel microRNAs in elongating cotton fiber cells.

BackgroundMicroRNAs (miRNAs) and other types of small regulatory RNAs play critical roles in the regulation of gene expression at the post-transcriptional level in plants. Cotton is one of the most economically important crops, but little is known about the roles of miRNAs during cotton fiber elongation.ResultsHere, we combined high-throughput sequencing with computational analysis to identify small RNAs (sRNAs) related to cotton fiber elongation in Gossypium hirsutum L. (G. hirsutum). The sequence analysis confirmed the expression of 79 known miRNA families in elongating fiber cells and identified 257 novel miRNAs, primarily derived from corresponding specific loci in the Gossypium raimondii Ulbr. (G. raimondii) genome. Furthermore, a comparison of the miRNAomes revealed that 46 miRNA families were differentially expressed throughout the elongation period. Importantly, the predicted and experimentally validated targets of eight miRNAs were associated with fiber elongation, with obvious functional relationships with calcium and auxin signal transduction, fatty acid metabolism, anthocyanin synthesis and the xylem tissue differentiation. Moreover, one tasiRNA was also identified, and its target, ARF4, was experimentally validated in vivo.ConclusionThis study not only facilitated the discovery of 257 novel low-abundance miRNAs in elongating cotton fiber cells but also revealed a potential regulatory network of nine sRNAs important for fiber elongation. The identification and characterization of miRNAs in elongating cotton fiber cells might promote the further study of fiber miRNA regulation mechanisms and provide insight into the importance of miRNAs in cotton.

Transcutaneous neuromuscular electrical stimulation can improve swallowing function in patients with dysphagia caused by non-stroke diseases: a meta-analysis.

There is still debate over whether the effect of transcutaneous neuromuscular electrical stimulation (NMES) in dysphagia rehabilitation is superior to traditional therapy (TT). The purpose of this meta-analysis was to assess the overall efficacy by comparing the two treatment protocols. Published medical studies in the English language were obtained by comprehensive searches of the Medline, Cochrane and EMBASE databases from January 1966 to December 2011. Studies that compared the efficacy of treatment and clinical outcomes of NMES versus TT in dysphagia rehabilitation were assessed. Two reviewers independently performed data extraction. Data assessing swallowing function improvement were extracted as scores on the Swallowing Function Scale as the change from baseline (change scores). Seven studies were eligible for inclusion, including 291 patients, 175 of whom received NMES and 116 of whom received TT. Of the seven studies, there were two randomised controlled trials, one multicentre randomised controlled trial and four clinical controlled trials. The change scores on the Swallowing Function Scale of patients with dysphagia treated with NMES were significantly higher compared with patients treated with TT [standardised mean difference (SMD) = 0·77, 95% confidence interval (CI): 0·13 to 1·41, P = 0·02]. However, subgroup analysis according to aetiology showed that there were no differences between NMES and TT in dysphagia post-stroke (SMD = 0·78, 95% CI: -0·22 to 1·78, P = 0·13, 4 studies, 175 patients). No studies reported complications of NMES. NMES is more effective for treatment of adult dysphagia patients of variable aetiologies than TT. However, in patients with dysphagia post-stroke, the effectiveness was comparable.

Molecular cloning and functional characterization of a DREB1/CBF-like gene ( GhDREB1L ) from cotton

The transcription factors DREB1s/CBFs play important roles in the regulation of plant resistance to environmental stresses and are quite useful for generating transgenic plants tolerant to these stresses. In the present work, a cDNA encoding DREB1/CBF-like protein (GhDREB1L) from cotton was isolated, and its sequence features, DNA binding preference, and expression patterns of the transcripts were also characterized. GhDREB1L contained one conserved AP2/ERF domain and its amino acid sequence was similar to the DREB1/CBF group of the DREB family from other plants. The DNA-binding domain of GhDREB1L was successfully expressed as a fusion protein in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Electrophoretic mobility shift assay revealed that the purified GhDREB1L fusion protein had a specific binding activity with the previously characterized DRE element (core sequence, ACCGAC) and also with the DRE-like sequence (core sequence, GCCGAC) in the promoter of the dehydration-responsive late embryogenesis-abundant gene LEA D113. Semi-quantitative RT-PCR showed that GhDREB1L was induced in the cotton cotyledons by low temperature, as well as drought and NaCl treatments. These results suggested that the novel cotton GhDREB1L might play an important role in response to low temperature as well as drought and high salinity through binding to the DRE cis-element.

Characterization of a Novel Annexin Gene from Cotton (Gossypium hirsutum cv CRI 35) and Antioxidative Role of its Recombinant Protein

Plant annexins represent a multigene family involved in cellular elongation and development. A cDNA encoding a novel annexin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library and designated GhAnx1. This gene encodes a 316 amino acid protein with a theoretical molecular mass of 36.06 kDa and a theoretical pI of 6.19. At the amino acid level, it shares high sequence similarity and has evolutionary relationships with annexins from higher plants. The purified recombinant protein expressed in Escherichia coli was used to investigate its physicochemical properties. Circular dichroism spectrum analyses showed a positive peak rising to the maximum at 196 nm and a broad negative band rounding 215 nm, suggesting that the GhAnx1 protein was prominently α-helical. The fluorescence measurements indicated that it could bind to Ca(2+) in vitro. These results demonstrated that GhAnx1 was a typical annexin protein in cotton. A bioassay experiment was conducted to analyze its potential function and showed that E. coli cells expressing GhAnx1 were protected from tert-butyl hydroperoxide (tBH) stress, suggesting that it had a potential antioxidative role. Northern blot analyses revealed that GhAnx1 was highly expressed in fibers, especially during the elongation stage, suggesting that it might be important for fiber elongation.

The cotton dehydration-responsive element binding protein GhDBP1 contains an EAR-motif and is involved in the defense response of Arabidopsis to salinity stress.

Our previous work reported that cotton dehydration-responsive element (DRE) binding protein 1 (GhDBP1) could function as an active transcriptional repressor for DRE-mediated gene expression. However, the repression mechanism utilised by GhDBP1 was unclear. In this report, we demonstrate that GhDBP1’s transcriptional repression domain is located at the C-terminus, and is known as an ERF-associated amphiphilic repression (EAR)-motif. Furthermore, the amino acid residues aspartic acid (D), leucine (L), asparagine (N) and proline (P) are conserved in the EAR-motif, and were found to be necessary for repression through mutational analysis. In addition, our promoter assays demonstrated a dehydration-induced and rehydration-repressed expression pattern of GhDBP1. Transgenic Arabidopsis plants overexpressing GhDBP1 were more sensitive to high salinity stress and appeared to downregulate the expression levels of the stress-induced effecter genes. Taken together, our findings provide an important insight into GhDBP1’s potential molecular repression mechanism and how it is involved in plant stress responses.