Wangbao Gong

Genome-wide identification and characterization of conserved and novel microRNAs in grass carp (Ctenopharyngodon idella) by deep sequencing

MicroRNAs (miRNAs) are post-transcriptional regulators which bind to target to regulate protein expression by repressing translation or promoting degradation of the target mRNA. Studies have shown that deep sequencing is a powerful tool for the identification of miRNAs, and it is believed that may more miRNAs remain to be discovered in grass carp. In the present study, a pool of equal amounts of RNA obtained from 8 different adult grass carp tissues (spleen, liver, muscle, kidney, skin, testis, gut and heart) was sequenced using deep sequencing technology. A total of 16.579.334 raw reads were yielded from the pooled small RNA library. Using bioinformatics analysis, we identified 160 conserved miRNAs and 18 novel miRNAs in grass carp. Randomly selected 6 conserved and 2 novel miRNAs were confirmed their expression by stem-loop qRT-PCR assay. Furthermore, the 1212 potential targets of these miRNAs were predicted using miRNA target prediction tool. GO and KEGG pathway enrichment analyses indicated relevant biological processes. Our study provides the first genome-wide investigation of miRNAs from 8 mixed tissues of grass carp, and the data obtained expand the known grass carp miRNA inventory and provide a basis for further understanding functions of grass carp miRNAs.

Identification of the conserved and novel microRNAs by deep sequencing and prediction of their targets in Topmouth culter

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play important roles in regulation of almost all biological processes in organisms. Topmouth culter is an economically important fish species in the Chinese freshwater polyculture system for its delicacy and high economic value. However, to date, no miRNAs have been reported in Topmouth culter species. In this study, the liver, muscle, spleen and kidney of Topmouth culter are sampled and used for construction of a small RNA library for deep sequencing. A total of 273 conserved miRNAs/miRNAs-star and 8 novel miRNAs/miRNAs-star were discovered, these miRNAs belong to 86 different miRNA families. There is a strong base bias on miRNA nucleotide compositions at certain positions. U is the dominant nucleotide, particularly at the beginning, middle, and end of conserved miRNAs. The expression of all 8 novel identified miRNAs in Topmouth culter was validated by using stem-loop qRT-PCR. Furthermore, the potential targets were predicted for these miRNAs, which were found to be involved in diverse biological processes. Our study is the first significantly enriches the Topmouth culter miRNA repertoire and provides a reference for further elucidation of complex miRNA-mediated regulatory networks for gene expression in the growth and developmental progression of Topmouth culter.

Gene Expression Profiling of Grass Carp (Ctenopharyngodon idellus) and Crisp Grass Carp.

Grass carp (Ctenopharyngodon idellus) is one of the most important freshwater fish that is native to China, and crisp grass carp is a kind of high value-added fishes which have higher muscle firmness. To investigate biological functions and possible signal transduction pathways that address muscle firmness increase of crisp grass carp, microarray analysis of 14,900 transcripts was performed. Compared with grass carp, 127 genes were upregulated and 114 genes were downregulated in crisp grass carp. Gene ontology (GO) analysis revealed 30 GOs of differentially expressed genes in crisp grass carp. And strong correlation with muscle firmness increase of crisp grass carp was found for these genes from differentiation of muscle fibers and deposition of ECM, and also glycolysis/gluconeogenesis pathway and calcium metabolism may contribute to muscle firmness increase. In addition, a number of genes with unknown functions may be related to muscle firmness, and these genes are still further explored. Overall, these results had been demonstrated to play important roles in clarifying the molecular mechanism of muscle firmness increase in crisp grass carp.

Cloning, mRNA expression and bioinformatic analysis of full length type collagen alpha 1 I cDNA from grass carp (Ctenopharyngodon idellus)

TypeⅠcollagen, as a number of collagen family, is the most abundant collagen and major components of the extracellular matrices of all metazoan life, and plays crucial roles in differentiation, formation of collagen fibers and tissue remodeling after injury, etc. Type Ⅰ collagen alpha 1(COL1A1) cDNA of grass carp (Ctenopharyngodon idella) was isolated through the RT-PCR and RACE approaches. The cDNA was 5 772-bp in length, including a 4 347-bp CDS (coding sequence) and encoded a polypeptide of 1 449 aa. The homology of COL1A1 amino acid with relative species (zebrafish, goldfish, etc.) was as high as 93% with zebra fish and goldfish. The protein peptide molecular weight was 137.2 ku and theoretical pI was 5.44 using ProtParam software on line. The protein peptides of COL1A1 possessed 6 α-helixes, 12 β-sheets, others of ruleless coil regions, and 18 regions of triple helical repeats, 22 low complexity regions, 17 function domains. There were two calcium-binding sites and one zinc-binding site in the COL1A1 protein peptide. COL1A1 mRNA was determined in all the tested 8 tissues (muscle, intestine, hepatopancreas, gill, skin, fin, kidney and spleen) of grass carp by semi- quantitative RT-PCR, and the mRNAs expression in gill, kidney, skin and fin significantly higher than other tissues (P0.05). The structure and bioinformatics characteristics of the COL1A1 from grass carp may help to further understand the function of COL1A1 gene in the repair process of damaged tissue in the grass carp.

Effect of C/N ratio on bioflocs formation and water quality in zero-water exchange grass crap tanks

Biofloc technology(BFT)was recently proposed as a new approach towards more sustainable practices that lower environmental impacts and prevent diseases incidence.In order to explore required adaptive C/N of bioflocs formation,the effects of C/N on bioflocs formation,water quality and growth performance of grass carp in zero-water exchange tanks were investigated.A locally formulated and prepared feed containing 30% crude protein with C/N ratio 10.8 was applied to all ponds.In order to raise the C/N ratio of the feed input to 15,20 and 25,glucose was applied separately as a source of carbohydrate in addition to artificial feed.The results showed that when the C/N was over 15,the bioflocs could effectively regulate water quality,reduce ammonia and nitrite concentration;the bioflocs volume index(FVI)was increasing with gradual increase in breeding time and stabilized on 14 d in each group;With the C/N increased,the crude protein content of bioflocs groups was significantly higher(P0.05)than that of control groups,but there was a decreasing trend in the growth of grass carp.Overall,when C/N was 15 in the biofloc technology used in the breeding of grass carp,this ratio could effectively promote the bioflocs formation and reduce ammonia nitrogen and nitrite nitrogen levels.

Analysis of bacterial community structure of Bio-Floc by PCR-DGGE

Bio-Floc consists of phytoplankton,bacteria,aggregates of living and dead particulate organic matter,and grazers of the bacteria.It not only can regulate water quality,but also can supply the food protein for breeding animals.For the study of bacterial community structure of Bio-Floc in freshwater culture systems,the key technologies of Israeli Avnimelech team about Bio-Floc in the field of aquaculture were used in this paper.Glucose was added as the carbon source which makes a C∶ N ratio of 20∶ 1 be maintained during the experiment.The bacterial communities of Bio-Floc were analyzed by using the PCR-DGGE(PCR-denaturing gradient gel electrophoresis)on the 5th,10th,and 15th day,respectively.The results indicated that the similarity of bacterial communities was highest on the 5th day and 10th day which was 67.4%,and was lowest on the 5th day and 15th day which was 40.5%.Diversity of bacterial species in the Bio-Floc was maximal on the 10th day and minimal on the 15 day.The acquired sequences of 24 bands in DGGE gel were performed by BLAST searches against NCBI database,and the sequences including the closely related sequences were aligned with Clustal W program in BioEdit software for phylogenetic analysis.The main microbes represented by 24 main bands in DGGE gel were Alphaproteobacteria,Gammaproteobacteria,Betaproteobacteria,Actinobacteria,Bacilli and Bacteroidetes.Among these bacteria,the α-,β-,and γ-proteobacteria are the major bacterial groups,but Actinobacteria bacteria exist on the 10th day and the 15th day.α-proteobacteria was the dominant bacteria during the entire process.The specific bacteria at different stages were usually the flocculant-producing bacteria:Acidovorax,Aeromonas,Agrobacterium only for 5th day;Bacillus,Rhodococcus for 10th day and 15th day,respectively.This study first found that the composition and diversity of bacteria of the Bio-Floc used in freshwater aquaculture systems are extremely rich.The study of functional microbial composition of the Bio-Floc may help to lay the foundation for the further application of the bio-floc technology in actual aquaculture production.

Denitrification potential evaluation of a newly indigenous aerobic denitrifier isolated from largemouth bass Micropterus salmoides culture pond

This work evaluates the application potential of a new indigenous aerobic denitrifier, strain Pseudomonas CW-2, isolated from a largemouth bass culture pond. The rate of ammonium-N removal by strain CW-2 was approximately 97% at a DO concentration of 5.2 mg/L. Furthermore, when nitrate and ammonia coexisted, the strain gave priority to assimilating ammonia, and thereafter to denitrification. Under optimal cultivation conditions, citrate and acetate were the carbon resources, C/N was 8, dissolved oxygen was 5.2 mg/L, and pH was 7; the removal rate of ammonium reached nearly 90%. The changing patterns of different bacteria in strain CW-2-treated and the control pond water were also compared. Lower levels of ammonia, nitrite, and phosphates were observed in the treated water as compared with the controls. Meanwhile, phylum-level distributions of the bacterial OTUs revealed that Proteobacteria, Bacteroidetes, Planctomycetes, and Nitrospirae continuously changed their relative abundances in relation to carbon and the addition of strain CW-2; this finding implies that the conventional denitrification process was weakened under the effects of carbon or the presence of strain CW-2. We propose that strain CW-2 is a promising organism for the removal of ammonium in intensive fish culture systems, according to our evaluations of its denitrification performance.

Identification and expression analysis of miRNA in hybrid snakehead by deep sequencing approach and their targets prediction

MicroRNAs (miRNAs) play important regulatory roles in numerous biological processes, but there is no report on miRNAs of hybrid snakehead. In this study, four independent small RNA libraries were constructed from the spleen, liver kidney and muscle of hybrid snakehead. These libraries were sequenced using deep sequencing technology, as result, a total of 1,067,172, 1,152,002, 1,653,941 and 970,866 clean reads from these four libraries were obtained. 252 known miRNAs and 63 putative novel miRNAs in these small RNA dataset were identified. The stem-loop RT-qPCR analysis indicated that eight known miRNAs and two novel miRNAs show different expression in eight different kinds of tissues. For better understanding the functions of miRNAs, 95,947 predicated target genes were analyzed by GO and their pathways, the results indicated that these targets of the identified miRNAs are involved in a broad range of physiological functions.

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.

Smad4-dependent regulation of type I collagen expression in the muscle of grass carp fed with faba bean

Smad4 is the key regulator in the transforming growth factor β1 (TGF-β1)/Smads signal pathway, and is also the crux of the regulation of type I collagen expression in mammals. In fish, however, the relationship between Smad4 and type I collagen is still unknown. Given the widely accepted importance of type I collagen in fish muscle hardness, we seek to explore this issue by analyzing the expressions of the TGF-β1/Smads pathway molecules and type I collagen in the muscle of crisp grass carp fed with faba bean, which shows increased muscle hardness. The study found that (1) in the process of feeding the grass carp with faba bean, the mRNA and protein expressions of TGF-β1, Smad2 and Smad4 all increased along with the increase of type I collagen expression (Col1α1 and Col1α2); (2) one day after the injection of Smad4 over-expression vector, both mRNA and protein expressions of Col1α1 and Col1α2 significantly increased, reaching the maximum on the 2nd and 5th day, respectively; (3) one day after the injection of Smad4 RNAi interference vector, the mRNA and protein expressions of Col1α1 and Col1α2 decreased, reaching the minimum on the 5th day. These results revealed that Smad4 is the major regulator of type I collagen in the muscle of grass carp fed with faba bean. This study would provide an important mechanistic basis for nutritional regulation of type I collagen in the muscle of fish.