A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on scre...A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control f gene expression in the gynogenetic fish embryogenesis.展开更多
The kinematics of turning maneuvers of startled Crucian Carp (Carassius auratus) are presented. All escape responses observed are C-type fast-starts. The position of the center of mass and the moment of inertia of the...The kinematics of turning maneuvers of startled Crucian Carp (Carassius auratus) are presented. All escape responses observed are C-type fast-starts. The position of the center of mass and the moment of inertia of the fish are calculated. The results show that the position of the center of mass is always at 35% of the length of the fish from the head and the position of the center of mass and moment of inertia can be considered unchanged during C-start of Crucian Carp. Hydrodynamic analysis of the C-start is given based on the kinematics data from our experiments. The C-start consists of three stages. In stage 1, the tail fin of fish rapidly flaps in one direction, and a large moment acts on the fish′s body, which rotates around the center of mass with an angular acceleration. In stage 2, the tail fin flaps more slowly in the opposite direction at slower speed, the fish′s body rotates around the center of mass with angular deceleration and the center of mass of the fish moves along an arc. In stage 3, the moment approximately equals zero, the fish′s body stops rotating and the center of mass the moves along a straight line.展开更多
The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimicti...The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp. MPF activity was measured by using histone H1 as phosphorylation substrate. There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes, but there existed some subtle difference between them. The subtle difference was that the first peak of MPF kinase activity was kept to a longerlasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp. It was suggested that the difference may be related to the spindle behavior changes, such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.展开更多
Background:Sperm abnormalities are one of the primary factors leading to male sterility,but their pathogenesis is still unclear.Although miRNAs are suggested to exert important roles in the regulation of spermatogenes...Background:Sperm abnormalities are one of the primary factors leading to male sterility,but their pathogenesis is still unclear.Although miRNAs are suggested to exert important roles in the regulation of spermatogenesis at both transcriptional and posttranscriptional levels,little is currently known regarding the regulation of sperm flagella assembly by microRNAs(miRNAs).The role of miRNAs in the development of sperm abnormalities in sterile triploid fish has not been studied.Results:In this study,we found that miR-199-5p was widely expressed in all detected tissues of different-ploidy crucian carp.As one of the testis-specific candidate markers,Tekt1 was predominantly expressed in the testis.Quantitative real-time PCR(qRT-PCR)analyses showed that the expression trend of miR-199-5p was exactly opposite to that of Tekt1.Through bioinformatics analysis,we identified a putative miR-199-5p binding site in the Tekt1 mRNA.We further identified Tekt1 as a target of miR-199-5p using luciferase reporter assay.Finally,we confirmed that miR-199-5p was necessary for sperm flagellar assembly and spermatogenesis in vivo via intraperitoneal injection of miR-199-5p antagomir or agomir in diploid red crucian carp.Moreover,miR-199-5p gainof-function could lead to spermatids apoptosis and abnormal spermatozoa structure,which is similar to that of allotriploid crucian carp.Conclusions:Our studies suggested that abnormally elevated miR-199-5p inhibited the sperm flagella formation in spermiogenesis by negatively regulating the expression of Tekt1,thereby causing sperm abnormalities of male allotriploid crucian carp.展开更多
[Objective] To investigate the effects of amino acid powder, fermented rapeseed meal and fermented cottonseed meal on the growth performance, amino acid absorption, body composition and liver function of allogynogenti...[Objective] To investigate the effects of amino acid powder, fermented rapeseed meal and fermented cottonseed meal on the growth performance, amino acid absorption, body composition and liver function of allogynogentic crucian carp. [ Method] The allogynogentic crucian carps with the average initial weight of 17.3 g were divided into control group and six experimental groups randomly. The allogynogentic crucian carps in the control group and experimental groups were fed a basal diet and the basal diets supplemented by amino acid powder, fermented rapeseed meal and fermented cottonseed meal, respectively. The growth rate and specific growth rate of allogynogentic crucian carps, the content of amino acids in serum and feeds, the composition indicators of body, muscle and liver, as well as the activities of glutamic-pyruvic transaminase （GPT） and glutamic-oxaloacetic transaminase （GOT） in liver and serum were determined, respectively. [ Result] The growth rate and specific growth rate （SGR） of allogynogentic crucian carps in the 3% amino acid powder group and the 6% fermented cottonseed meal group were increased greatly （ P 〈 0.05）, and no significant difference was found between experimental group and control group. The serum lysine content of the 3% amino acid powder group and the 6% fermented cottonseed meal group was higher than that of the control group. In the experimental groups, the absorption of four essential amino acids was up to a peak at 4 h after feeding, which was consistent with the control group. The body composition and liver function of allogynogentic crucian carps had no significant difference between the experimental groups and the control group. [ Conclusion] The 3% amino acid powder, 6% fermented cottonseed meal and 6% fermented rapeseed meal can substitute fish meal, cottonseed meal and rapeseed meal to improve the clrowth performance, amino acid absorption of the allogynogentic crucian carps.展开更多
The interest in the analysis of phospholipids(PLs), especially phosphatidylcholine(PC), has been increasing due to the importance of them in biochemistry as well as in industry. A method was reported based on the offl...The interest in the analysis of phospholipids(PLs), especially phosphatidylcholine(PC), has been increasing due to the importance of them in biochemistry as well as in industry. A method was reported based on the offline combination of MALDI-TOF MS and normal-phase HPLC for analyzing PLs extracted from crucian carp. Total PLs of crucian carp were extracted and then separated by HPLC before the collected subfractions were analyzed by MALDI-TOF MS. The mass spectra obtained show peaks of H+, Na+ and K+ adducts of PC molecules. It is shown that the prior separation of PLs by HPLC is highly necessary to remove the signal suppressing and to avoid the possible overlapping. With this method, 9 possible PC molecules in crucian carp and the corresponding fatty acid compositions were given from the well-resolved mass spectra.展开更多
Marinate the clams in brine for 3 hours, until silt isdiffused, and keep them fresh. Draw the crucian carp,and make 3 or 4 diagonal cuts on the surface of bothsides.
Crucian carp(Carassius auratus gibelio),an extensively cultivated freshwater fish,was one of the model species for the study of fish immunology.Polyclonal antibodies were advantageous molecular tools for studying tele...Crucian carp(Carassius auratus gibelio),an extensively cultivated freshwater fish,was one of the model species for the study of fish immunology.Polyclonal antibodies were advantageous molecular tools for studying teleost immune system.Specifically,polyclonal antibodies reacting with immunoglobulins(Ig)were used successfully in studies of the teleost fishes.In the present study,we produced polyclonal antibody against CH2 domains of crucian carp IgM,and measured the in vivo dynamics of IgM mRNA caused by CyHV-2 infection.The recombinant protein IgM with relative molecular weight about 53 KD was correctly expressed in prokaryotic cells.The specificity of the polyclonal antibody was evaluated by Western blotting and results revealed that the antibody not only specifically recognized crucian carp serum but also cross-reacted with grass carp serum.Quantitative RT-PCR analysis demonstrated the expression of IgM mRNA changed significantly after CyHV-2 infection.The expression of IgM in the kidney increased and reached a maximum at 6 h post-infection(hpi),while dropped to a low level at 5 days post-infection(dpi).In conclusion,the expression of IgM was significantly upregulated in the kidney of crucian carp infected with CyHV-2,indicating that IgM played a potential role in systemic immunity against viral infection.Polyclonal antibody against crucian carp IgM had certain clinical relevance,which might provide insight into the early stage of virus infection and prevention of the disease.展开更多
The recombinant plasmid pFV2cfGH was constructed using Growth hormone gene which was cloned from the channel catfish cDNA library,the β-action promoter of carp and the polyadenylation signal from the salmon growth ho...The recombinant plasmid pFV2cfGH was constructed using Growth hormone gene which was cloned from the channel catfish cDNA library,the β-action promoter of carp and the polyadenylation signal from the salmon growth hormone gene, and recombinant plasmid pFV2cfGH was microinjected into the fertilized eggs of the colorful crucian carp. 60 transgenic Colorful Crucian carps total DNA were got ,and growth hormone gene were detected by PCR. The result shows that the channel catfish growth hormone gene had been transmitted into the genome of the Colorful Crucian carp and the integration rate is about 47%.展开更多
基金This work was supported by the National Nat-ural Science Foundation of China(Grant No.30130240),the Chinese Academy of Sciences(GrantNo.KSCX2-SW-303).
文摘A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control f gene expression in the gynogenetic fish embryogenesis.
基金This work was supported by the National Natural Science Foundation(No.10332040,10072063)the Innovation Project of the Chinese Academy of Sciences(No.KJCX—SW-L04).
文摘The kinematics of turning maneuvers of startled Crucian Carp (Carassius auratus) are presented. All escape responses observed are C-type fast-starts. The position of the center of mass and the moment of inertia of the fish are calculated. The results show that the position of the center of mass is always at 35% of the length of the fish from the head and the position of the center of mass and moment of inertia can be considered unchanged during C-start of Crucian Carp. Hydrodynamic analysis of the C-start is given based on the kinematics data from our experiments. The C-start consists of three stages. In stage 1, the tail fin of fish rapidly flaps in one direction, and a large moment acts on the fish′s body, which rotates around the center of mass with an angular acceleration. In stage 2, the tail fin flaps more slowly in the opposite direction at slower speed, the fish′s body rotates around the center of mass with angular deceleration and the center of mass of the fish moves along an arc. In stage 3, the moment approximately equals zero, the fish′s body stops rotating and the center of mass the moves along a straight line.
文摘The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp. MPF activity was measured by using histone H1 as phosphorylation substrate. There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes, but there existed some subtle difference between them. The subtle difference was that the first peak of MPF kinase activity was kept to a longerlasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp. It was suggested that the difference may be related to the spindle behavior changes, such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.
基金supported by the National Natural Science Foundation of China(Grant No.31872551)Laboratory of Lingnan Modern Agriculture Project(Grant No.NT2021008)+4 种基金the Natural Science Foundation of Hunan Province for Distinguished Young Scholars(Grant No.2020JJ2022)Hunan Provincial Innovation Foundation for Postgraduate(Grant No.CX20200480)the earmarked fund for China Agriculture Research System(Grant No.CARS-45)the Key Research and Development Program of Hunan Province(Grant No.2020NK2016)111 Project(D20007).
文摘Background:Sperm abnormalities are one of the primary factors leading to male sterility,but their pathogenesis is still unclear.Although miRNAs are suggested to exert important roles in the regulation of spermatogenesis at both transcriptional and posttranscriptional levels,little is currently known regarding the regulation of sperm flagella assembly by microRNAs(miRNAs).The role of miRNAs in the development of sperm abnormalities in sterile triploid fish has not been studied.Results:In this study,we found that miR-199-5p was widely expressed in all detected tissues of different-ploidy crucian carp.As one of the testis-specific candidate markers,Tekt1 was predominantly expressed in the testis.Quantitative real-time PCR(qRT-PCR)analyses showed that the expression trend of miR-199-5p was exactly opposite to that of Tekt1.Through bioinformatics analysis,we identified a putative miR-199-5p binding site in the Tekt1 mRNA.We further identified Tekt1 as a target of miR-199-5p using luciferase reporter assay.Finally,we confirmed that miR-199-5p was necessary for sperm flagellar assembly and spermatogenesis in vivo via intraperitoneal injection of miR-199-5p antagomir or agomir in diploid red crucian carp.Moreover,miR-199-5p gainof-function could lead to spermatids apoptosis and abnormal spermatozoa structure,which is similar to that of allotriploid crucian carp.Conclusions:Our studies suggested that abnormally elevated miR-199-5p inhibited the sperm flagella formation in spermiogenesis by negatively regulating the expression of Tekt1,thereby causing sperm abnormalities of male allotriploid crucian carp.
基金supported by the Applied Basic Research Projects of Suzhou City (YJG0912)
文摘[Objective] To investigate the effects of amino acid powder, fermented rapeseed meal and fermented cottonseed meal on the growth performance, amino acid absorption, body composition and liver function of allogynogentic crucian carp. [ Method] The allogynogentic crucian carps with the average initial weight of 17.3 g were divided into control group and six experimental groups randomly. The allogynogentic crucian carps in the control group and experimental groups were fed a basal diet and the basal diets supplemented by amino acid powder, fermented rapeseed meal and fermented cottonseed meal, respectively. The growth rate and specific growth rate of allogynogentic crucian carps, the content of amino acids in serum and feeds, the composition indicators of body, muscle and liver, as well as the activities of glutamic-pyruvic transaminase （GPT） and glutamic-oxaloacetic transaminase （GOT） in liver and serum were determined, respectively. [ Result] The growth rate and specific growth rate （SGR） of allogynogentic crucian carps in the 3% amino acid powder group and the 6% fermented cottonseed meal group were increased greatly （ P 〈 0.05）, and no significant difference was found between experimental group and control group. The serum lysine content of the 3% amino acid powder group and the 6% fermented cottonseed meal group was higher than that of the control group. In the experimental groups, the absorption of four essential amino acids was up to a peak at 4 h after feeding, which was consistent with the control group. The body composition and liver function of allogynogentic crucian carps had no significant difference between the experimental groups and the control group. [ Conclusion] The 3% amino acid powder, 6% fermented cottonseed meal and 6% fermented rapeseed meal can substitute fish meal, cottonseed meal and rapeseed meal to improve the clrowth performance, amino acid absorption of the allogynogentic crucian carps.
基金Supported by the Knowledge Innovation Program of the Chinese Academy of Sciences(No.KSCX2-YW-N-035)Projects of Shenyang City Science and Technology Burea, China(Nos.F10-224-4-00, 1091239-6-00)
文摘The interest in the analysis of phospholipids(PLs), especially phosphatidylcholine(PC), has been increasing due to the importance of them in biochemistry as well as in industry. A method was reported based on the offline combination of MALDI-TOF MS and normal-phase HPLC for analyzing PLs extracted from crucian carp. Total PLs of crucian carp were extracted and then separated by HPLC before the collected subfractions were analyzed by MALDI-TOF MS. The mass spectra obtained show peaks of H+, Na+ and K+ adducts of PC molecules. It is shown that the prior separation of PLs by HPLC is highly necessary to remove the signal suppressing and to avoid the possible overlapping. With this method, 9 possible PC molecules in crucian carp and the corresponding fatty acid compositions were given from the well-resolved mass spectra.
基金Supported by National Natural Science Foundation of China (No.30571414,No 30640015)Changsha Municipal Project of Science and Technology (No.K051127-72).
文摘Marinate the clams in brine for 3 hours, until silt isdiffused, and keep them fresh. Draw the crucian carp,and make 3 or 4 diagonal cuts on the surface of bothsides.
基金supported by the General Fund Project of China Shanghai Natural Science Foundation(Program No.22ZR1427200).
文摘Crucian carp(Carassius auratus gibelio),an extensively cultivated freshwater fish,was one of the model species for the study of fish immunology.Polyclonal antibodies were advantageous molecular tools for studying teleost immune system.Specifically,polyclonal antibodies reacting with immunoglobulins(Ig)were used successfully in studies of the teleost fishes.In the present study,we produced polyclonal antibody against CH2 domains of crucian carp IgM,and measured the in vivo dynamics of IgM mRNA caused by CyHV-2 infection.The recombinant protein IgM with relative molecular weight about 53 KD was correctly expressed in prokaryotic cells.The specificity of the polyclonal antibody was evaluated by Western blotting and results revealed that the antibody not only specifically recognized crucian carp serum but also cross-reacted with grass carp serum.Quantitative RT-PCR analysis demonstrated the expression of IgM mRNA changed significantly after CyHV-2 infection.The expression of IgM in the kidney increased and reached a maximum at 6 h post-infection(hpi),while dropped to a low level at 5 days post-infection(dpi).In conclusion,the expression of IgM was significantly upregulated in the kidney of crucian carp infected with CyHV-2,indicating that IgM played a potential role in systemic immunity against viral infection.Polyclonal antibody against crucian carp IgM had certain clinical relevance,which might provide insight into the early stage of virus infection and prevention of the disease.
文摘The recombinant plasmid pFV2cfGH was constructed using Growth hormone gene which was cloned from the channel catfish cDNA library,the β-action promoter of carp and the polyadenylation signal from the salmon growth hormone gene, and recombinant plasmid pFV2cfGH was microinjected into the fertilized eggs of the colorful crucian carp. 60 transgenic Colorful Crucian carps total DNA were got ,and growth hormone gene were detected by PCR. The result shows that the channel catfish growth hormone gene had been transmitted into the genome of the Colorful Crucian carp and the integration rate is about 47%.
