Investigation of a sex pheromone for the oriental cockroach,Blatta orientalis

A sex pheromone for adult male oriental cockroaches Blatta orientalis was isolated from the faeces of adult virgin female oriental cockroaches. It elicited a sexual response at 10 pg and 1 ng with B. orientalis and Periplaneta americana adult males, respectively. The site of production appers to be the crop, oesophagus, and proventriculus. Electroantennogram responses of male antennae toward the isolated pheromone were greater than those of the female antennae. The adult male oriental cockroach also responded to the American cockroach sex pheromone. The isolated pheromone with a mol. wt of 232 may be similar to one of the components of the American cockroach sex pheromone.     

Rapid cloning and bioinformatic analysis of spinach Y chromosome-specific EST sequences

The genome of spinach single chromosome complement is about 1000 Mbp, which is the model material to study the molecular mechanisms of plant sex differentiation. The cytological study showed that the biggest spinach chromosome (chromosome 1) was taken as spinach sex chromosome. It had three alleles of sex-related X, X ^m and Y. Many researchers have been trying to clone the sex-determining genes and investigated the molecular mechanism of spinach sex differentiation. However, there are no successful cloned reports about these genes. A new technology combining chromosome microdissection with hybridization-specific amplification (HSA) was adopted. The spinach Y chromosome degenerate oligonucleotide primed-PCR (DOP-PCR) products were hybridized with cDNA of the male spinach flowers in florescence. The female spinach genome was taken as blocker and cDNA library specifically expressed in Y chromosome was constructed. Moreover, expressed sequence tag (EST) sequences in cDNA library were cloned, sequenced and bioinformatics was analysed. There were 63 valid EST sequences obtained in this study. The fragment size was between 53 and 486 bp. BLASTn homologous alignment indicated that 12 EST sequences had homologous sequences of nucleic acids, the rest were new sequences. BLASTx homologous alignment indicated that 16 EST sequences had homologous protein-encoding nucleic acid sequence. The spinach Y chromosome-specific EST sequences laid the foundation for cloning the functional genes, specifically expressed in spinach Y chromosome. Meanwhile, the establishment of the technology system in the research provided a reference for rapid cloning of other biological sex chromosome-specific EST sequences.     

Identification and comparative analysis of the ovary and testis microRNAome of mud crab Scylla paramamosain

The role of microRNA (miRNA) in reproductive regulation is attracting increasingly more attention. In this study, we obtained 9,643,114 and 15,498,999 raw reads from the ovary and testis library of important farmed mud crab Scylla paramamosain, respectively. After data mining, a total of 4,096,464 and 11,737,973 mappable small RNA sequences remained for analysis. By mapping to the reference genome and expressed sequence tag (EST) of Daphnia pulex and other crabs, a total of 1,417 miRNAs were identified. On the basis of 1,417 miRNAs, 514 (36.3%) unique miRNAs coexpressed in the gonad of female and male libraries, and 336 (23.7%) and 567 (40%) expressed preferentially in female and male libraries, respectively. Analysis of library sequencing data resulted in the identi?cation of 108 miRNAs (out of 1,417; 7.6%) that showed signi?cant differential expression between the two samples. Of these, 13 miRNAs were expressed only in the testis, two miRNAs were expressed only in the ovary, and 93 miRNAs were coexpressed: 57 (61.3%) were upregulated (ovary/testis) and 36 (38.7%) were downregulated (ovary/testis). To confirm the expression patterns of the predicted miRNAs, we randomly selected 14 candidate miRNAs from 108 differentially expressed miRNAs and performed stem–loop real time quantitative PCR (RT‐qPCR) assays in five ovary developing stages. Five miRNAs showed similar expression patterns in almost every stage as those revealed by identification of differentially expressed genes (IDEG6) analysis. The above five miRNAs were predicted to match the 3′‐untranslated region of the published S. paramamosain gene. Four out of five miRNA had a regulation effect on many genes, especially the genes related to gonadal development.     

Identification and validation of Asteraceae miRNAs by the expressed sequence tag analysis

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a vital role in the regulation of gene expression. Despite their identification in hundreds of plant species, few miRNAs have been identified in the Asteraceae, a large family that comprises approximately one tenth of all flowering plants. In this study, we used the expressed sequence tag (EST) analysis to identify potential conserved miRNAs and their putative target genes in the Asteraceae. We applied quantitative Real-Time PCR (qRT-PCR) to confirm the expression of eight potential miRNAs in Carthamus tinctorius and Helianthus annuus. We also performed qRT-PCR analysis to investigate the differential expression pattern of five newly identified miRNAs during five different cotyledon growth stages in safflower. Using these methods, we successfully identified and characterized 151 potentially conserved miRNAs, belonging to 26 miRNA families, in 11 genus of Asteraceae. EST analysis predicted that the newly identified conserved Asteraceae miRNAs target 130 total protein-coding ESTs in sunflower and safflower, as well as 433 additional target genes in other plant species. We experimentally confirmed the existence of seven predicted miRNAs, (miR156, miR159, miR160, miR162, miR166, miR396, and miR398) in safflower and sunflower seedlings. We also observed that five out of eight miRNAs are differentially expressed during cotyledon development. Our results indicate that miRNAs may be involved in the regulation of gene expression during seed germination and the formation of the cotyledons in the Asteraceae. The findings of this study might ultimately help in the understanding of miRNA-mediated gene regulation in important crop species.     

高通量测序技术分析急性髓系白血病血浆外泌体微RNA表达谱差异

急性髓系白血病（acute myeloid leukemia,AML）是一类造血干细胞的恶性克隆性疾病,目前的诊断方法不利于疾病的早期发现,且诊断结果重复性较差。已有大量研究显示,细胞外microRNA（miRNA或miR）富集在外泌体（exosome）中,且受其表面膜的保护而具有很好的稳定性,是理想的分子标志物。目前,多种实体肿瘤均已检测到肿瘤特异性外泌体miRNA(exosomal miRNA)。然而,在AML患者中未见此外泌体miRNA报道。本研究探讨急性髓系白血病血浆外泌体miRNA表达谱差异及新miRNA序列。采用solexa高通量测序技术对7例AML患者（AML组）及7例健康对照者（对照组）血浆外泌体miRNAs进行测序,利用Mireap预测软件进行新miRNAs分析,通过edger差异分析软件筛选组间差异miRNA,获得211个已知的差异表达miRNAs以及2个新miRNAs,选择4个差异表达的miRNAs：miR-155-5p、miR-335-5p、miR-451a及xxx-m0038 5p（新miRNA）,在两组（各23例）的血浆外泌体样本中,进行实时荧光定量PCR（qRT-PCR）验证,验证结果与测序结果一致。对差异表达的外泌体miRNA进行靶基因预测及其GO（Gene Ontology）和信号通路富集分析,发现靶基因聚集的生物学功能多数参与生物进展过程的调控。靶基因主要富集在FoxO、MAPK、Hippo信号通路以及HTLV-I感染等。结果显示,AML患者与健康对照者的血浆外泌体miRNA存在着差异性表达。差异性表达的miRNA特异性很高,对进一步阐明AML白血病发生与发展的分子机制、研发新的无创诊断方法、新的诊断标记物和有效治疗AML的方法具有十分重要和深远的意义。     

Comparative Analysis and Identification of miRNAs and Their Target Genes Responsive to Salt Stress in Diploid and Tetraploid Paulownia fortunei Seedlings

Salt stress is a global environmental problem that affects plant growth and development. Paulownia fortunei is an adaptable and fast-growing deciduous tree native to China that is environmentally and economically important. MicroRNAs (miRNAs) play important regulatory roles in growth, development, and stress responses in plants. MiRNAs that respond to biotic stresses have been identified; however, how miRNAs in P. fortunei respond to salt stress has not yet been reported. To identify salt-stress-responsive miRNAs and predict their target genes, four small RNA and four degradome libraries were constructed from NaCl-treated and NaCl-free leaves of P. fortunei seedlings. The results indicated that salt stress had different physiological effects on diploid and tetraploid P. fortunei. We detected 53 conserved miRNAs belonging to 17 miRNA families and 134 novel miRNAs in P. fortunei. Comparing their expression levels in diploid and tetraploid P. fortunei, we found 10 conserved and 10 novel miRNAs that were significantly differentially expressed under salt treatment, among them eight were identified as miRNAs probably associated with higher salt tolerance in tetraploid P. fortunei than in diploid P. fortunei. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to predict the functions of the target genes of the conserved and novel miRNAs. The expressions of 10 differentially expressed miRNAs were validated by quantitative real-time polymerase chain reaction (qRT-PCR). This is the first report on P. fortunei miRNAs and their target genes under salt stress. The results provided information at the physiological and molecular levels for further research into the response mechanisms of P. fortunei to salt stress.     

Identification and Comparative Analysis of the Tegillarca granosa Haemocytes MicroRNA Transcriptome in Response to Cd Using a Deep Sequencing Approach

Background

MicroRNAs (miRNAs) are endogenous non-coding small RNAs (sRNAs) that can base pair with their target mRNAs, which represses their translation or induces their degradation in various biological processes. To identify miRNAs regulated by heavy metal stress, we constructed two sRNA libraries for the blood clam Tegillarca granosa: one for organisms exposed to toxic levels of cadmium (Cd) and one for a control group.

Results

Sequencing of the two libraries and subsequent analysis revealed 215 conserved and 39 new miRNAs. Most of the new miRNAs in T. granosa were up- or down-regulated in response to Cd exposure. There were significant differences in expression between the Cd and control groups for 16 miRNAs. Of these, five miRNAs were significantly up-regulated and 11 were significantly down-regulated in the Cd stress library. Potential targets were predicted for the 16 differential miRNAs in pre-miRNAs identified according to sequence homology. Some of the predicted miRNA targets are associated with regulation of the response to stress induced by heavy metals. Five differentially expressed miRNAs (Tgr-nmiR-8, Tgr-nmiR-21, Tgr-miR-2a, Tgr-miR-10a-5p, and Tgr-miR-184b) were validated by qRT-PCR.

Conclusion

Our study is the first large-scale identification of miRNAs in T. granosa haemocytes. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in the responses of this species to environmental heavy metal stresses.     

Identification and comparative analysis of the miRNA expression profiles from four tissues of Micropterus salmoides using deep sequencing

In the present study, four small RNA libraries were constructed from an M. salmoides population and sequenced using deep sequencing technology. A total of 9,888,822; 8,519,365; 20,566,198; and 15,762,254 raw reads representing 666,097; 755,711; 978,923; and 840,175 unique sequences were obtained from the spleen, liver, kidney, and muscle libraries, respectively. As a result, 509 known miRNAs belonging to 143 families and 1157 novel miRNAs were identified. The miRNAs displayed diverse expression levels among the four libraries, among which most of the known miRNAs were expressed at higher levels than the novel miRNAs. Furthermore, stem-loop qRT-PCR was applied to validate and profile the expression of the differentially expressed miRNAs in the four different tissues, which revealed that some miRNAs showed tissue specific expression. The identification of miRNAs in M. salmoides will provide new information and enhance our understanding of the functions of miRNAs in regulating biological processes.