Transgenic Yellow River carp is characterized by rapid growth rate and high feed-conversion efficiency and exhibits a great application prospect. However, there is still a significant separation of growth traits in the transgenic Yellow River carp family; as such, growth-related genotypes must be screened for molecular marker-assisted selection. In this study, 23 growth-related candidate genes containing 48 SNP markers were screened through bulked segregant analysis (BSA) among transgenic Yellow River carp family members showing significant separation of growth traits. Then, two growth-related genes (Nos. 17 and 14 genes) were identified through combined genome-wide association study (GWAS) of candidate genes and validation of the full-sibling family approach. Nos. 17 and 14 genes encode BR serine/threonine-protein kinase 2 (BRSK2) and eukaryotic translation-initiation factor 2-alpha kinase 3 (Eif2ak3), respectively. The average body weight of three subgroups carrying the genotypes 17GG, 17GG+14CC, and 17GG+14TT of these two genes increased by 27.96, 38.28, and 33.72%, respectively, compared with the controls. The proportion of individuals with body weight >500g in these subgroups increased by 19.22, 26.82, and 30.92%, respectively. The results showed that appropriate genotype carriers can be selected from the progeny population through BSA sequencing combined with simplified GWAS analysis. Hence, basic population for breeding can be constructed and transgenic Yellow River carp strains with stable production performance and uniform phenotypic properties can be bred.=20
Growth and reproduction are closely related. Growth hormone (GH)-transgenic common carp exhibit accelerated growth and delayed reproductive development, which provides an amenable model to study hormone cross talk between the growth and reproductive axes. We analyzed the energy status and reproductive development in GH-transgenic common carp by using multi-tissue RNA sequencing, real-time-PCR, Western blotting, ELISA, immunofluorescence, and in vitro incubation. The expression of gys (glycogen synthase) and igfbp1 (insulin-like growth factor binding protein) as well as blood glucose concentrations are lower in GH-transgenic carp. Agrp1 (agouti-related protein 1) and sla (somatolactin a), which are related to appetite and lipid catabolism, are significantly higher in GH-transgenic carp. Low glucose content and increased appetite indicate disrupted metabolic and energy deprivation status in GH-transgenic carp. Meanwhile, the expression of genes, such as gnrhr2 (gonadotropin-releasing hormone receptor 2), gtha (gonadotropin hormone, alpha polypeptide), fsh beta (follicle stimulating hormone, beta polypeptide), lh beta [luteinizing hormone, beta polypeptide] in the pituitary, cyp19a1a (aromatase A) in the gonad, and cyp19a1b (aromatase B) in the hypothalamus, are decreased in GH-transgenic carp. In contrast, pituitary gnih (gonadotropin inhibitory hormone), drd1 (dopamine receptor D1), drd3 (dopamine receptor D3), and drd4 (dopamine receptor D4) exhibit increased expression, which were associated with the retarded reproductive development. Leptin receptor mRNA was detected by fluorescence in situ hybridization in the pituitary including the pars intermedia and proximal pars distalis, suggesting a direct effect of leptin on LH. Recombinant carp Leptin protein was shown to stimulate pituitary gtha, fsh beta, lh beta expression, and ovarian germinal vesicle breakdown in vitro. In addition to neuroendocrine factors, we suggest that reduced hepatic leptin signaling to the pituitary might be part of the response to overexpression of GH and the resulting delay in puberty onset.
Integrin beta-1 (ITGB1) is a transmembrane protein belonging to the integrin family and it plays an important role in viral entry. In this study, the itgb1b gene of the rare minnow, Gobiocypris rarus, was cloned and analyzed. To investigate the possible role of itgb1b on grass carp reovirus (GCRV) infection, we generated an ITGB1b-deficient rare minnow (ITGB1b-/-) using the CRISPR/Cas9 system. Following stimulation with GCRV, the survival time of the -ITGB1b-/- rare minnows was extended in comparison to the wild-type minnows. Moreover, the relative copy number of GCRV and the level of clathrin-mediated endocytosis-associated and apoptosis-related gene expression in the ITGB1b-/- rare minnows was significantly lower than that of the wild-type minnows. These results suggested that the absence of itgb1b reduced viral entry efficiency and the expression of apoptosis-related genes. Moreover, the data suggested that itgb1b played an important role in mediating the entry of viruses into the cells via clathrin. Therefore, these findings provide novel insight into the function of itgb1b in the process of GCRV infection.=20
Su, Jianguo
Zhu, Zuoyan
Wang, Yaping
Xiong, Feng
Zou, Jun
The ability to utilize the RNA interference (RNAi) machinery for silencing target-gene expression has created a lot of excitement in the research community. In the present study, we used a cytomegalovirus (CMV) promoter-driven DNA template approach to induce short hairpin RNA (shRNA) triggered RNAi to block exogenous Enhanced Green Fluorescent Protein (EGFP) and endogenous No Tail (NTL) gene expressions. We constructed three plasmids, pCMV-EGFP-CMV-shGFP-SV40, pCMV-EGFP-CMV-shNTL-SV40, and pCMV-EGFP-CMV-shScrambled-SV40, each containing a CMV promoter driving an EGFP reporter cDNA and DNA coding for one shRNA under the control of another CMV promoter. The three shRNA-generating plasmids and pCMV-EGFP control plasmid were introduced into zebrafish embryos by microinjection. Samples were collected at 48 h after injection. Results were evaluated by phenotype observation and real-time fluorescent quantitative reverse-transcription polymerase chain reaction (Q-PCR). The shGFP-generating plasmid significantly inhibited the EGFP expression viewed under fluorescent microscope and reduced by 70.05 +/- 1.26% of exogenous EGFP gene mRNA levels compared with controls by Q-PCR. The shRNA targeting endogenous NTL gene resulted in obvious NTL phenotype of 30 +/- 4% and decreased the level of their corresponding mRNAs up to 54.52 +/- 2.05% compared with nontargeting control shRNA. These data proved the feasibility of the CMV promoter-driven shRNA expression technique to be used to inhibit exogenous and endogenous gene expressions in zebrafish in vivo.
The common carp, Cyprinus carpio, is the third most cultivated freshwater species worldwide, but is also considered an invasive species. The Trojan Y chromosome strategy is one of the most promising methods to eradicate this invasive species. However, obtaining fertile YY supermale (MYY) and YY physiological female (FYY) common carp was thought to be very difficult. The present study focused on the production of androgenetic MYY and XY physiological female (FXY) common carp. We first optimized the conditions for artificially induced androgenesis. The results indicated that the optimum ultraviolet (UV) exposure time was 4min at an irradiation distance of 26cm and the optimum initiation time with heat shock (40 +/- 0.5C for 2min) was 30min after fertilization. Then, we produced androgenetic MYY Yellow River carp with only paternal inheritance, which were viable and identified by paternity testing and test crossing. Finally, we successfully produced FXY Yellow River carp by feeding 60-d-old MXY with commercially available feed mixed with 17-estradiol (200mg/kg) and Flutamide (200mg/kg) for 3mo. In conclusion, by combining artificially induced androgenesis with an artificially induced sex reversal technique, we could cost-effectively produce MYY and FXY common carp.
Su, Jianguo
Zhu, Zuoyan
Wang, Yaping
Zou, Jun
Wang, Na
Jang, Songhun
Dicer catalyzes the initiation step of RNA interference (RNAi) which is known to play a significant role in innate immune response to viral infection in many organisms. To study the RNAi-related pathway after virus infection in fish, we identified a partial cDNA sequence of dicer from rare minnow, Gobiocypris rants. Real-time quantitative RT-PCR (qRT-PCR) demonstrated the Dicer transcript level was the highest at zygote stage, decreased at prim-5 stage, and was stable from the protruding mouth to adult stage. Regular RT-PCR analysis showed that the Dicer gene expressed widely in the tested tissues, including brain, gill, heart, intestine, kidney, liver, muscle, ovary, spleen and testis. The expression of Dicer mRNA was significantly increased in the early period of Grass carp reovirus (GCRV) infection, and declined from 24 It post-injection (h p.i.) (P<0.05). The mRNA expression returned to control levels at 48 h p.i. (P>0.05). Under transmission electron microscope, virions were difficulty to find out in 12 h p.i., and virus inclusion bodies and few scattered viral particles were easily visualized from 24 h p.i. to moribund. These results implied GCRV triggered the RNAi pathway in the early stages of infection and perhaps virus inclusion bodies suppressed the antiviral functions of RNAi mechanism. (C) 2009 Published by Elsevier B.V.
Wang, Xiangming
Liu, Min
Li, Weida
Suh, Christopher D.
Zhu, Zuoyan
Jin, Yishi
Fan, Qichang
Background: The serine/threonine kinase BUB1 (Budding Uninhibited by Benzimidazole 1) was originally identified in yeast as a checkpoint protein, based on its mutant's incapacity of delaying the cell cycle in response to loss of microtubules. Our understanding of its function is primarily from studies carried out in yeast S. cerevisiae. It has been shown that it is a component of the mitotic spindle checkpoint and regulates the separation of sister chromatids through its downstream molecules. However, its roles in multi-cellular organisms remain unclear. Methods and Findings: In nematode C. elegans, rapid cell divisions primarily occur in embryos and in germline of postembryonic larvae and adults. In addition, a select set of cells undergo a few rounds of cell division postembryonically. One common phenotype associated with impaired cell division is described as Stu (Sterile and Uncoordinated) [1,2]. We conducted a genetic screen for zygotic mutants that displayed Stu phenotype in C. elegans. We isolated seven Stu mutants that fell into five complementation groups. We report here that two mutations, FanWang5 (fw5) and FanWang8 (fw8) affect the bub-1 gene, a homolog of yeast BUB1. Both mutant alleles of fw5 and fw8 exhibited variable behavioral defects, including developmental arrest, uncoordination and sterility. The number of postembryonically born neurons in the ventral cord decreased and their axon morphology was abnormal. Also, the decrease of neurons in the ventral cord phenotype could not be suppressed by a caspase-3 loss-of-function mutant. In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages. We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans. Studies in yeast have shown that BUB1 functions as a spindle checkpoint protein by regulating the anaphase promoting complex/cyclosome (APC/C). We performed double mutant analysis and observed that bub-1 genetically interacted with several downstream genes, including fzy-1/CDC20, mat-2/APC1 and emb-27/APC6. Conclusions: Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically. Further, our genetic analysis is consistent with that the function of bub-1 in C. elegans is likely similar to its yeast and mammalian homologs.
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