Schizothorax argentatus that only distributes in the Ili River basin in Xinjiang is one of the rare and endangered species of schizothorax in China, thus has high scientific and economic values. In this study, the complete mitochondrial genome sequence of S. argenteus with a length of 16 580 bp was obtained by high-throughput sequencing. The gene compositions and arrangement were similar to those of typical vertebrates. It contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a non-coding region (D-loop). The nucleotide compositions were A (30.25%), G (17.28%), C (27.20%), and T (25.27%), respectively, showing obvious AT bias and anti-G bias. Among the tRNA genes, only tRNA-Ser^(GCU) could not form a typical cloverleaf structure due to the lack of dihydrouracil arm. The AT-skew and GC-skew values of the ND6 gene were fluctuating the most, suggesting that the gene may experience different selection and mutation pressures from other genes. The mitochondrial control region of S. argenteus contained three different domains, i.e., termination sequence region (ETAS), central conserved region (CSB-F, CSB-E, CSB-D, and CSB-B), and conserved sequence region (CSB1, CSB2, and CSB3). The conserved sequence fragment TT (AT) nGTG, which was ubiquitous in Cypriniformes, was identified at about 50 bp downstream CSB3. Phylogenetic relationships based on the complete mitochondrial genome sequence of 28 Schizothorax species showed that S. argenteus had differentiated earlier and had a distant relationship with other species, which may be closely related to the geographical location and the hydrological environment where it lives.
Animals ; Genome, Mitochondrial/genetics* ; Phylogeny ; Sequence Analysis, DNA ; Cyprinidae/genetics* ; RNA, Transfer/genetics* ; DNA, Mitochondrial/genetics* ; Genes, Mitochondrial

The sequence variation of medicinal fish of Culter (Pisces: Cyprinidae) was analyzed by using cytochrome c oxidase subunit I (COI) sequencing collected from different regions of the Yangtze River basin, and we examine whether barcoding of COI can be used to discriminate medicinal fish of Culter. The AT content in the COI region of medicinal fish of Culter was higher than that of GC, which was similar with other species of Cypriniformes. Ninty-six percent of nucleotide changes were observed at the 3rd codon position of COI sequence, but the amino acid compositions translated by COI sequences of all Culter fish stayed the same. It is suggested that most synonymous mutations might occur at the 3rd position. The average Kimura-2-parameter (K2P) distance within-species was lower than 1%, and the K2P distance of pairwise-species was 10 times as much as that of within-species. The phylogenetic tree estimated by Neighbour-joining method indicated that species within genera invariably clustered, and generally so did individuals within species. Individuals from operational taxonomic units designated as different Culter species, supporting morphological evidence for each of these being separate species. It is suggested that the COI barcoding can be used to identify medicinal fish species of Culter.
Animals ; China ; Cyprinidae ; classification ; genetics ; DNA Barcoding, Taxonomic ; DNA, Mitochondrial ; genetics ; Electron Transport Complex IV ; genetics ; Fish Proteins ; genetics ; Medicine, Chinese Traditional ; Molecular Sequence Data ; Phylogeny

In this study, pEGFP-N1 was chosen as the reporter plasmid and transferred into Ctenopharyngodon idellus kidney (CIK) cells by electroporation, and the optimal electroporation conditions were determined by testing the transfection efficiency with different voltages, pulse times, plasmid amounts, and numbers of shocks. The results showed that the maximum electroporation efficiency was achieved under the following conditions in a 0.2 cm electroporation cuvette containing CIK cells (1.5 x 10(7)/mL, 200 microl): electric voltage 200 V, pulse time 45 ms, plasmid 30 microg, and one electric shock. The total genomic RNA of grass carp reovirus (GCRV) was extracted in this experiment and reversely transcribed into cDNA, which was used to amplify the gene segment of GCRV non-structural protein NS26 using designed specific primers. The PCR product was recombined into pEGFP-N1 vector. The fusion protein EGFP-NS26 was successfully and efficiently expressed in the CIK cells by electroporation, which was confirmed by both fluorescent imaging and Western blot analysis. This experiment laid a foundation for further functional studies of the non-structural protein NS26 of GCRV.
Animals ; Cell Line ; Cyprinidae ; Electroporation ; Fish Diseases ; virology ; Gene Expression ; Kidney ; virology ; Reoviridae ; genetics ; physiology ; Reoviridae Infections ; veterinary ; virology ; Viral Nonstructural Proteins ; genetics ; metabolism

Through PCR amplification, 1.3 kb of 5'-proximal promoter (TA, 1.3 kb) of the beta-actin gene of white cloud mountain minnow Tanichthys albonubes was obtained. Using Genome Walker, a 1.7 kb 5'-upstream sequence from the proximal promoter of the beta-actin gene was isolated, and a further promoter (3.0 kb in size) was amplified according to the isolated 5'-proximal and upstream sequences (TLA, 3.0 kb). Both the 1.3 kb and 3.0 kb promoters contain elements that were critical to the transcription activity of other species, including the CCAAT Box (-89 approximately -85), CArG Box (-59 approximately -49), TATA Box (-26 approximately -20). Results of putative transcription binding sites analysis of the promoters by software TRANSFAC 6.0 revealed the presence of E-box, several transcript binding sites NF-Y, SP1 (Stimulating Protein 1), AP1 (Activator Protein 1), and some more transcription binding sites existing in the further promoter. The two promoter sequences were inserted into the expression vector to construct the recombinant expression vector, pTA-DsRed and pTLA-DsRed, respectively. The vectors were microinjected into the fertilized eggs of Tanichthys albonubes and higher positive rate was obtained and stronger red fluorescence was observed in pTLA-DsRed transgenic fish. RT-PCR analysis showed that RFP (Red fluorescent protein) mRNA level in pTLA-DsRed transgenic fish was 35.7% higher than that of the pTA-DsRed transgenic fish of 15-days-post-hatched. The present study showed that both the proximal and further promoter sequences have effective transcription activities and the 3.0 kb promoter possesses higher potent activity than that of the 1.3 kb promoter.
5' Untranslated Regions ; genetics ; Actins ; genetics ; metabolism ; Animals ; Animals, Genetically Modified ; genetics ; Base Sequence ; Cyprinidae ; genetics ; Genetic Vectors ; Molecular Sequence Data ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism