Structural conservation and food habit-related liver expression of uncoupling protein 2 gene in five major Chinese carps

The full-length cDNA of grass carp (Ctenopharyngodon idellus) and silver carp (Hypophthalmichthys molitrix) uncoupling protein 2 (UCP2) was obtained from liver. The grass carp UCP2 cDNA was determined to be 1152 bp in length with an open reading frame that encodes 310 amino acids. Five introns (Intron 3, 4, 5, 6 and 7) in the translated region, and partial sequence of Intron 2 in the untranslated region of grass carp UCP2 gene were also obtained. Gene structure comparison between grass carp and mammalian (human and mouse) UCP2 gene shows that, the UCP2 gene structure of grass carp is much similar to that of human and mouse. Partial UCP2 cDNA sequences of bighead carp (Aristichthys nobilis) and mud carp (Cirrhinus molitorella), were further determined. Together with the common carp (Cyprinus carpio) UCP2 sequence from GenBank (AJ243486), multiple alignment result shows that the nucleotide and amino acid sequences of the UCP2 gene, were highly conserved among the five major Chinese carps that belong to four subfamilies. Using beta-actin as control, the ratio UCP2/beta-actin mRNA (%) was determined to be 149.4 +/- 15.6 (common carp), 127.4 +/- 22.1(mud carp), 96.7 +/- 12.7 (silver carp), 94.1 +/- 26.8 (bighead carp) and 63.7 +/- 16.2 (grass carp). The relative liver UCP2 expression of the five major Chinese carps, shows a close relationship with their food habit: benthos and detritus-eating fish (common carp and mud carp) > planktivorious fish (silver carp and bighead carp) > herbivorous fish (grass carp). We suggest that liver UCP2 might be important for Chinese carps to detoxify cyanotoxins and bacteria in debris and plankton food.     

鲤鱼肌肉生长抑制素基因(MSTN)的克隆及其组织表达特征

肌肉生长抑制素(Myostatin,MSTN)是动物肌肉发育和生长过程中的负调控因子,对MSTN的研究将有助于促进动物生产。鲤鱼是我国的主要淡水养殖对象之一。因此,我们采用RT-PCR方法克隆了鲤鱼MSTN cDNA(No.EF551058)的部分序列,长度为921bp,编码306个氨基酸残基。鲤鱼MSTN具有MSTN的共同特征,有蛋白酶水解位点RIRR和9个保守的半胱氨酸残基。多重序列比较发现其与斑马鱼GDF8有极近的亲缘关系,96.7%的氨基酸序列同源。不同组织的RT-PCR分析发现鲤鱼MSTN主要在肌肉和脑部表达,而其他所检测组织未见表达。鲤鱼MSTN不仅在肌肉生长发育中发挥作用,可能在神经系统发育中也有其作用。     

黄颡鱼MSTN基因多态性及其与生长性状的相关性分析

肌肉生长抑制素基因(Myostatin,MSTN)属于转化生长因子β家族,其主要功能是负向调控肌肉的生长发育。采用PCR-SSCP技术对黄颡鱼MSTN基因进行单核苷酸多态性检测和分型,并与其生长性状进行关联分析。结果表明,在第一内含子部分检测到1个缺失位点和2个突变位点(T1003del、G1022A和T1063G),基因型分别为:AA、AB、CC、CD和DD;在第三外显子部分检测到1个突变位点(T132C),基因型分别为:EE和EF。关联性分析表明,AA基因型个体的全长、体长、体高、体厚、头长和体重显著大于CD和DD基因型(P<0.05),AA基因型雌性个体的全长、体长、体高、体厚、头长、尾柄高、尾柄厚和体重也显著大于DD基因型(P<0.05)。由此推断,AA基因型是影响雌性黄颡鱼生长性状的有利基因型,DD基因型是影响雌性黄颡鱼生长性状的不利基因型,可以尝试利用这两个位点对雌性黄颡鱼进行标记辅助选育。     

草鱼生肌因子5基因的克隆及其组织表达谱分析

目的:获得草鱼生肌因子5(Myf-5)基因序列,分析其在草鱼不同组织和不同发育阶段中的表达规律。方法:根据鲤鱼Myf-5基因序列设计引物,用草鱼肌肉组织总RNA,经RT-PCR扩增其Myf-5基因序列;利用半定量RT-PCR分析草鱼Myf-5基因在草鱼不同组织和不同发育阶段的mRNA表达特性。结果:获得了草鱼Myf-5基因开放读框序列723 bp,GenBank登录号为GU290227;该基因编码由240个氨基酸残基组成的蛋白,具有MyoD家族基因的典型性碱性螺旋-环-螺旋(bHLH)结构,其氨基酸序列与斑马鱼、鲤鱼、虹鳟、大西洋鲑等的同源性较高(74%~97%),与哺乳动物和禽类如人、小鼠、大鼠、猪、牛和鸡的同源性较低(56%~60%);在草鱼红肌、白肌、肝胰脏、肾脏、脑和肠中均检测到Myf-5基因的表达,红肌、白肌和脑组织中Myf-5基因mRNA的表达量显著高于其他组织(P<0.05);草鱼Myf-5基因的表达随着其生长发育呈下降趋势,在较大规格试验鱼(500 g)中的表达显著低于其他2种规格(50~60 g、120~130 g)的试验鱼(P<0.05)。结论:获得了草鱼Myf-5基因序列,其在红肌、白肌和脑组织中的表达量显著高于其他组织,并随生长发育呈下降趋势,为研究Myf-5在草鱼肌肉发育过程中的作用提供了基础资料。     

SNPs in the myostatin gene of the mollusk Chlamys farreri: Association with growth traits

Myostatin (MSTN) is a member of the transforming growth factor-β superfamily which negatively regulates growth of muscle tissue. In this study, 103 cultivated Chlamys farreri individuals were screened for polymorphisms in the MSTN gene using PCR-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods. Two mutations were found: A/G at position 327 in exon 2, which caused an amino acid change from Thr to Ala (Thr305Ala), and C/T at position 289 in exon 3, which caused an amino acid change from Cys to Arg (Cys422Arg). One way ANOVA of the SNPs and growth traits showed that genotype GG of primer M5 had significantly higher body mass, soft-tissue mass, adductor muscle mass, shell length, shell height, absolute growth rate of shell height and body mass than those of genotype AG and AA (P < 0.05). Genotype frequencies of genotype AA, AG and GG were 68.94%, 27.18% and 3.88%, respectively. The results present evidence that the C. farreri MSTN gene may be selected as a candidate gene for these growth traits.     

Molecular Cloning,Identification, and Expression Patterns of Myostatin Gene in Water Buffalo (Bubalus Bubalis)

Myostatin (MSTN), also named growth differentiation factor 8 (GDF8), is a transforming growth factor-β (TGF-β) family member with a key role in the negative regulation of skeletal muscle growth. However, its role in ovarian folliculogenesis remains unclear. To provide us with a basis for understanding this role, we cloned MSTN and examined its expression patterns in water buffalo (Bubalus bubalis). The complete ORF of the water buffalo MSTN gene is 1,128 nucleotides, which encode a 375 amino acid protein and sharing 99% identity at the deducted amino acid level with that of Bos taurus. Protein sequence analysis showed that MSTN is a weakly acerbic extracellular protein, consisting of signal peptides at 18-19 sites, a TGF-β propeptide, and a TGF-β domain. RT-PCR analyses demonstrated that water buffalo MSTN was expressed in multiple tissues but not limited to muscle. Immunohistochemistry staining confirmed the presence of MSTN in oocytes and granulosal cells. To our knowledge, this is the first study to confirm the expression of MSTN in the water buffalo ovary, suggesting an additional role of MSTN in water buffalo folliculogenesis, along with its role in skeletal muscle growth regulation. Further study of the regulatory mechanism of MSTN in water buffalo reproduction is warranted.

Abbreviations: MSTN, myostatin; ORF, open reading frame.     

Myostatin (MSTN) gene duplications in Atlantic salmon (Salmo salar): evidence for different selective pressure on teleost MSTN-1 and -2

Whereas the negative muscle regulator myostatin (MSTN) in mammals is almost exclusively expressed in the muscle by a single encoding gene, teleost fish possess at least two MSTN genes which are differentially expressed in both muscular and non-muscular tissues. Duplicated MSTN-1 genes have previously been identified in the tetraploid salmonid genome. From Atlantic salmon we succeeded in isolating the paralogous genes of MSTN-2, which shared about 70% identity with MSTN-1a and -1b. The salmon MSTN-2a cDNA encoded a predicted protein of 363 residues and included the conserved C-terminal bioactive domain. MSTN-2a seemed to be primarily expressed in the brain, and a functional role of teleost MSTN-2 in the neurogenesis similar to the inhibitory action of the closely related GDF-11 in the mammalian brain was proposed. In contrast, a frame-shift mutation in exon 1 of salmon MSTN-2b would lead to the synthesis of a putatively non-functional truncated protein. The absence of processed MSTN-2b mRNA in the examined tissues indicated that this gene has become a non-functional pseudogene. The differential, but partially overlapping, expression patterns of salmon MSTN-2a, -1a and -1b in muscular and non-muscular tissues are probably due to the different arrangement of the potential cis-acting regulatory elements identified in their putative promoter regions. Single and paired E-boxes in the MSTN-1b promoter were shown to bind both homo-and hetero-dimers of the myogenic regulatory factor MyoD and E47 in vitro of importance for initiating the myogenic program. Analyses of nucleotide substitution patterns indicated that the teleost MSTNs essentially have evolved under purifying selection, but a subset of amino acid sites under positive selective pressure were identified within the MSTN1 branch. The results may reflect the evolutionary forces related to adoption of the different functional roles proposed for the teleost MSTN isoforms. The phylogenetic analysis of multiple vertebrate MSTNs suggested at least two separate gene duplication events in the fish lineage. Linkage analysis of polymorphic microsatellites within intron 2 of salmon MSTN-1a and -1b mapped the two genes to different linkage groups in agreement with the tetraploid origin of the duplicated salmonid MSTN-1 and MSTN-2 genes.     

Temporal and spatial expression pattern of the myostatin gene during larval and juvenile stages of the Chilean flounder (Paralichthys adspersus)

The full length cDNA sequence of the myostatin gene was cloned from a teleostean fish, the Chilean flounder (Paralichthys adspersus) through RT-PCR amplification coupled with the RACE approach to complete the 5'- and 3'-region. The deduced amino acid sequence encodes a protein of 377 amino acid residues, including the structural domains responsible for its biological activity. Amino acid sequence comparison revealed high sequence conservation, and confirmed that the isolated sequence corresponds to the MSTN1 gene. Gene expression analysis showed that cfMSTN mRNA is present in a wide variety of tissues in juvenile fish. In addition, we assessed the spatial expression pattern of the MSTN mRNA during embryos and larval stages through whole mount in situ hybridization. No expression was observed in embryos, whereas in larvae of 8 and 9 days post fertilization, the notochord, somites, intestine and some discrete territories in the head, such as brain and eye, were positive for MSTN mRNA. Our results contribute to the knowledge of the MSTN system in larval and juvenile stages; in particular the strong expression observed in the notochord suggests that MSTN, in synchronization with positive growth signals, may play an important role in the control of the development of larvae somites.     

草鱼IRE1-like基因的克隆、组织表达及其对微囊藻毒素-LR的响应

为了研究内质网应激相关基因需肌醇酶1(Inositol-requiring enzyme 1, IRE1-like)的结构和生物学功能及其在草鱼(Ctenopharyngodon idella)响应微囊藻毒素-LR(MC-LR)中的作用, 研究根据草鱼转录组测序结果得到该基因家族成员IRE1-like的EST序列, 采用RACE技术获得了草鱼IRE1-like基因的cDNA全长序列(登录号: MG797683)。该基因序列全长3595 bp, 包括3093 bp开放阅读框, 编码1030个氨基酸, 分子量为116.24 kD, 理论等电点为6.26, 具有跨膜结构和信号肽。草鱼IRE1-like基因包含Luminal (39—307 aa)、STKc (569—834 aa)和RNase (837—915 aa)三个结构域。同源性和系统进化树结果表明草鱼IRE1-like基因与斑马鱼IRE1-α基因亲缘关系最近。荧光定量PCR(qRT-PCR)检测结果表明, 草鱼IRE1-like基因在肝脏组织中的表达量最高, 在肌肉、脾脏、鳃和心脏等其他8种不同组织中也均有表达。不同剂量MC-LR诱导草鱼24h和96h后, 25 μg MC-LR/kg BW和100 μg MC-LR/kg BW剂量组中草鱼肝脏IRE1-like基因表达水平分别在24h和96h显著上升(P<0.05)。以上结果初步说明了草鱼IRE1-like基因可能在MC-LR诱导草鱼内质网应激中起着重要的作用, 为进一步研究草鱼IRE1-like基因的功能奠定了基础。     

NPY和POMC基因在生长差异与饥饿再摄食鳙个体中的表达分析

为研究摄食促进基因和摄食抑制基因对鱼类生长调控和饥饿再摄食过程的影响,研究克隆了鳙神经肽Y(HynNPY)基因和前阿黑皮素原(HynPOMC)基因cDNA序列,采用qRT-PCR技术分析它们在生长显著差异鳙个体下丘脑、肠中的基因表达变化;设置对照组(连续投喂4周)、饥饿组、饥饿再摄食组,分析NPY和POMC在不同处理组鳙的下丘脑、肠中的基因表达变化。鳙NPY和POMC基因cDNA全长分别为839和799 bp,开放阅读框有291和657 bp,分别编码96和218个氨基酸。系统进化分析结果表明,鳙NPY和POCM基因具有高度保守性。鳙NPY在下丘脑的表达量最高,其次为肠和脑; POMC在肠道中的表达量最高,其次为下丘脑和肝脏。在相同环境下生长差异鳙个体的下丘脑和肠中, NPY在极大个体的表达量高于极小组个体, POMC在极小个体中的表达量高于极大个体。饥饿导致NPY在下丘脑表达上升,在肠表达量显著上升,恢复摄食后, NPY在下丘脑和肠中表达量下降; POMC在饥饿组下丘脑和肠中都表现为表达量呈显著上升,复投喂后POMC表达量逐步下降至接近对照组水平。肠组织学观察显示,极大个体的肠腔直径...     

银鲫肌酸激酶M3-CK cDNA的克隆及其表达特征

用抑制性差减杂交结合SMART cDNA合成和RACE—PCR技术克隆到雌核发育银鲫(Carassius auratus gibelio)肌酸激酶M3-CK基因的全长cDNA。银鲫M3-CK cDNA全长1551bp，编码380个氨基酸，与普通鲤鱼(cyprinus carpio)M3-CK的氨基酸序列同源性高达95％。种系分析表明，银鲫M3-CK与其它脊椎动物的肌肉型肌酸激酶聚为较近的一支，与鲤鱼的M3-CK聚在一起，与脑特异型肌酸激酶及线粒体型肌酸激酶分歧较大。虚拟Northern杂交显示银鲫M3-CK基因在胚胎发育中差异表达。RT—PCR表明，银鲫M3-CK基因在成熟卵母细胞和胚胎发育早期可检测到少量的转录产物，在胚胎发育期间从肌肉效应期开始转录，并一直持续表达。组织RT—PCR表明，银鲫M3-CK基因只在心脏和肌肉表达。     

Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.     

鳙鱼同工酶发育遗传学研究

采用淀粉或聚丙烯酰胺凝胶电泳法分析鳙鱼早期发育阶段(从未受精卵到卵黄吸尽期)及成体不同组织(脑、眼、心、肌、肾、肝)中六种同工酶(LDH,MDH,IDH,ADH,SDH,EST)的分化表达模式。鳙鱼同工酶基因的表达具有明显的组织特异性。早期发育阶段,ADH和SDH均无染色活性;LDH、MDH和IDH具有不同的发育变化谱式,而EST酶谱在整个早期发育阶段均无明显变化。与鲢、草鱼相比,鳙鱼早期发育过程中胚胎Ldh-A基因激活的时间被推迟。上述结果可为鳙鱼种群的生化遗传结构分析以及鳙鱼的人工育种提供基础资料。     

净化时间对微流水系统中鳙品质影响的研究

以商品规格的鳙为研究对象, 利用湖泊建立为流水系统, 研究净化时间对系统中鳙肌肉的营养成分、质构和持水特性及肌肉中滋味成分与挥发性气味物质的影响。结果表明, 在整个净化过程, 鳙肌肉中蛋白、脂肪含量呈显著下降, 灰分含量处理前后无显著差异; 鳙肌肉中挥发性盐基氮含量、羰基含量随净化时间延长而显著下降; 肌肉质构的硬度随净化时间延长而显著上升, 黏着性和咀嚼性优于净化初期, 弹性和回复性与初期显示差异不显著; 水溶性蛋白含量和游离氨基酸含量呈先上升后下降的趋势。综上所述, 微流水净化处理能够显著提升鳙肌肉品质, 改善鳙肌肉口感和风味。     

鳙的线粒体基因组核苷酸全序列分析

对采集自我国长江的鳙的线粒体DNA全序列进行了测定.结果表明,鳙的线粒体DNA全长为166221 bp,其碱基因组成为A=31.6%;C=27.1%;G=16.0%;T=25.3%,A+T含量为56.9%.鳙线粒体基因组的排列、结构和组成与其它鲤科鱼类相似,包括37个基因,即13个蛋白质编码基因,2个rRNA基因,22个tRNA基因和一个非编码控制区(D-loop).在13个蛋白编码基因中,除ND6由轻链编码外,其余12个基因均由重链编码.COI基因的起始密码子为GTG,而其它12个蛋白编码基因的起始密码子均为ATG.