Molecular Cloning and Single Nucleotide Polymorphisms Identification of Myostatin cDNA in the Pearl Oyster,Pinctada fucata

Myostatin (MSTN or growth differentiation factor‐8) is considered a negative regulator of muscle growth and development. In this study, we cloned and characterized the full‐length MSTN cDNA from Pinctada fucata, and named it as the Pf‐MSTN cDNA. The single nucleotide polymorphisms (SNPs) in Pf‐MSTN cDNA were then screened and genotyped. The full‐length Pf‐MSTN cDNA was 2644 bp, including an open reading frame of 1248 bp encoding 415 amino acids which contained typical structural characteristics shared by all members of the transforming growth factor‐β (TGF‐β) superfamily including an N‐terminal signal peptide, a propeptide domain, and a TGF‐β superfamily bioactive domain. The Pf‐MSTN mRNA was detected in all tested tissues, with the highest mRNA levels observed in the adductor muscle, indicating that Pf‐MSTN may play a major role in this tissue. Furthermore, by sequencing and alignment, 32 SNP loci were identified in Pf‐MSTN cDNA. Genotyping 50 individuals from a common breeding stock revealed that 21 of these 32 loci were polymorphic. The minor allele frequency was in the range of 0.0400–0.4800, and the polymorphism information content value varied from 0.0739 to 0.3750. The observed and expected heterozygosity ranged from 0.0200 to 1.0000 and from 0.0776 to 0.5051, respectively. These SNPs identified in Pf‐MSTN will be useful for future studies investigating their utility in marker‐assisted selection for P. fucata breeding.     

缩骨鲫MSTN基因的克隆与组织表达分析

采用同源克隆、生物信息学方法及荧光定量PCR(Real-time PCR)对缩骨鲫(Carassius auratus sogu var.)肌肉生长抑制素(myostatin,MSTN)基因进行克隆,分析和组织表达状况研究。结果显示:缩骨鲫基因组MSTN基因全长4 737 bp,含有两个内含子和三个外显子;编码区长度为1 128 bp,编码375个氨基酸,包括信号肽(1aa~23aa),TGF-β前肽保守区域(34aa~256aa),TGF-β功能区(281aa~375aa),保守的RXXR蛋白酶酶切位点以及C-端活性区域9个保守的半胱氨酸残基,这与其他物种MSTN相似。此外,缩骨鲫与鲫鱼(Carassius auratus)的MSTN氨基酸序列同源性最高,达到96.1%。以β-actin为内参基因,荧光定量PCR分析表明,MSTN在肌肉中表达量最高;脑、眼、心脏中次之;在肝胰脏、卵巢、肠和肾脏中微量表达。     

加州鲈肌肉生长抑制素(MSTN)cDNA的克隆和序列分析

肌肉生长抑制素是抑制肌肉生长和发育的生长调控因子。对运用RT-PCR和RACE技术从加州鲈成鱼肌肉总RNA中扩增得到的MSTN cDNA全序列进行了序列分析。结果表明,加州鲈MSTN cDNA全长为1626bp,其开放阅读框为1 134bp,共编码377个氨基酸,前面的22个氨基酸为信号肽,中间有四个氨基酸(RARR)为蛋白水解加工位点;该基因总共有13个半胱氨酸残基,后面9个在蛋白水解加工位点之后的C端生物活性区,与其它脊椎动物比较,它们的位置完全一致,对该基因的结构和功能非常重要。与GenBank中已知的条纹狼鲈、金鲷、斑马鱼、虹鳟、斑点叉尾鮰、人、猪、鸡、鸽MSTN的ORF相比较,核苷酸序列同源性为63%~94.4%,氨基酸同源性为61.4%~96%,特别是在C端生物活性区氨基酸同源性为88.1%~100%,高度的保守性反映了该基因受到了高度的进化限制以及功能的重要性。加州鲈MSTN基因的克隆为研究该基因打靶和鱼类肌肉发育调控机理奠定了基础。     

岩原鲤MSTN基因克隆及饥饿对MSTN表达的影响

运用同源克隆的方法获得岩原鲤(Procypris rabaudi(Tchang))肌肉生长抑制素(myostatin,MSTN)c DNA全长序列,并通过逆转录PCR(RT-PCR)检测MSTN基因的组织差异性表达以及饥饿胁迫对MSTN基因表达的影响。结果显示,岩原鲤MSTN基因1 547 bp的c DNA序列,编码区为1 128 bp,编码375个氨基酸。PCR检测显示,该基因在肌肉和脑组织中大量表达,在眼、肠、心、鳃、肾和头肾组织中少量表达,在肝胰脏、脾脏组织中未见表达。饥饿再投喂实验表明肌肉组织中MSTN基因表达含量随着饥饿时间的延长逐渐升高,恢复投喂后3d表达含量急剧下降,投饵后6 d升至正常水平。结果表明,MSTN的表达对营养摄入敏感,在饥饿状态下岩原鲤通过上调MSTM表达来抑制肌肉生长,节约能量消耗。     

吉富罗非鱼 MSTN 基因结构及其多态性与生长性状的相关性

通过PCR和基因组步移法,从吉富罗非鱼DNA中扩增出肌细胞生长抑制素(MSTN)基因及其5′调控区。该序列全长2413bp,含有3个外显子,2个内含子。5′调控区462bp,外显子Ⅰ379bp,外显子Ⅱ371bp,部分外显子Ⅲ145bp,内含子Ⅰ305bp,内含子Ⅱ751bp,编码区共编码298个氨基酸。5′调控区含有与肌肉特异性基因转录密切相关的转录调控元件E-box以及其他一些转录调控元件,如TATAbox,OCT1,AP1,AP4。通过随机测序法寻找SNPs(Signal nucleotide poly morphisms),获得了3个SNPs,但在群体筛选中,只有内含子Ⅱ内的1个SNPs表现多态性。同时测量了96尾(45♂,51♀)吉富罗非鱼的体质量、体长、体高、体厚,并将这些数据与MSTN基因的SNPs多态性进行相关性分析,研究发现MSTN基因内含子Ⅱ的728nt处G/T多态与吉富罗非鱼体型(体厚/体长、体高/体长)存在显著相关(P0.05)。这些研究结果表明,MSTN基因的SNPs可作为吉富罗非鱼育种的候选分子标记。     

瘤背石磺肌肉生长抑制素基因的克隆及组织表达分析

为探究石磺由海洋向陆地进化过程中肌肉生长和发育的分子机制,实验以石磺科贝类转录组数据为基础,采用RACE方法从瘤背石磺肌肉中首次克隆到MSTN cDNA的全长并做了相应的组织表达分析。结果显示,瘤背石磺MSTN基因cDNA全长2667 bp,包括1650 bp的开放阅读框,374 bp的5′端非翻译区,643 bp的3'端非翻译区,共编码549个氨基酸。预测该基因编码的蛋白质原子总量为8776,分子式为C2774H4331N783O862S26,分子质量约为63.27 ku,理论等电点为6.02,信号肽预测结果显示,N端具有21个氨基酸长度的信号肽。瘤背石磺MSTN具有MSTN的共同特征,包括蛋白酶水解位点RSRR和C端多肽生物活性区以及9个保守的半胱氨酸残基。通过进化树分析,瘤背石磺MSTN与加州海兔MSTN的亲缘关系最近。RT-PCR结果显示,MSTN基因在各个组织中均有表达;含肌纤维组织中的表达量低于内脏器官的表达量,在肝胰腺中的表达量最高,腹足表达量最低。MSTN基因一级结构具有很高的保守性,说明该基因在进化上的限制性和功能的重要性;同时该基因在石磺非肌肉组织中表达,表明该基因不仅有抑制肌肉生长的作用,还参与其他生命活动的调节。     

A homologue of dermatopontin from Haliotis diversicolor and its response to pathogenic infection

Dermatopontin (DPT), a component of the extracellular matrix, plays important roles in cell‐matrix interactions and matrix assembly. Some studies have revealed that it has more general functions in biological activities. However, its function in molluscs is poorly understood. In this study, a molluscan DPT gene, saDPT2, was cloned from small abalone Haliotis diversicolor. The full‐length cDNA of saDPT2 sequence is 620 bp, with a 531 bp open reading frame encoding a protein of 177 amino acids (aa). Amino acid sequence analysis revealed that saDPT2 shares conserved signature motifs with other DPT proteins, including three repeats of 10‐residue motif (S‐X‐H‐X‐N‐X‐Y‐E‐D‐R), which is similar to mammalian 6‐residue repeating sequence of D‐R‐E/Q‐W‐X‐F/Y. Quantitative real‐time PCR was employed to investigate the tissue distribution of saDPT2 mRNA, its expression at different developmental stages, and in abalone under bacteria challenge. The saDPT2 mRNA could be detected in all examined tissues and developmental stages. Moreover, the saDPT2 mRNA was up‐regulated in haemocytes and gills after bacteria injection. The results indicate that the saDPT2 could respond to pathogenic infection and may play a role in adult abalone immune system.     

ENU诱变草鱼家系生长特性及肌肉生长抑制素基因SNP筛选的研究

经过175 d养殖,对4个N-乙基-N-亚硝基脲(ENU)诱变草鱼家系进行生长对比,采用显著性比较,偏相关分析和因子分析统计方法对草鱼体质量、全长、体长、头长、体高、尾柄长、尾柄高、体厚性状进行分析,进而运用双向测序法对MSTN1、MSTN2基因在具有明显差异家系中进行SNP位点筛选。试验结果显示,家系4的生长速度明显大于家系3,家系4的8个性状明显大于其他3个家系;家系1中体长、尾柄长、体厚与体质量密切相关,家系2中体长、头长、体厚与体质量密切相关,家系3中全长、体长、尾柄高与体质量密切相关,家系4中全长、体长、体厚与体质量密切相关。由此可知,家系4和家系3具有明显的生长差异。对于MSTN1基因,家系3在465位点C/G、467位点G/A,家系4在465位点C/G均发生错义突变;对于MSTN2基因,家系3和4在912位点C/T均发生同义突变,家系3在1027位点G/A、家系4在366位点A/G均产生错义突变,位于非编码区1390位点A/T和1401位点G/A在两个家系均发现SNP位点。试验结果表明,MSTN1、MSTN2基因的不同SNP位点与ENU诱变草鱼的生长性状存在紧密联系。     

牙鲆肌肉生长抑素(MSTN)基因克隆

采用同源克隆及基因组步移的方法，分离克隆了牙鲆肌肉生长抑素（MSTN）基因。经过序列分析及cDNA验证，牙鲆MSTN基因具有3个外显子和2个内含子，编码377个氨基酸。5`侧翼区含有8个TATA框，一个CAAT框，6个E框；3`侧翼区含有加尾信号。通过同源分析，牙鲆MSTN C末端含有9个保守半胱氨酸残基和一个RVRR蛋白酶酶切位点；通过进化树分析，牙鲆MSTN与鱼类MSTN基因聚为一支。RT-PCR分析表明，牙鲆MSTN在胚胎发育中不表达或表达量较低，说明MSTN在牙鲆胚胎发育中并不起重要作用；其在各组织中的表达，随个体和环境的不同而有差异，暗示MSTN的表达受外界因素调控。     

Targeted Sequencing of Myogenic Regulatory Factors and Myostatins Reveals an Association between MSTN‐1 and Interorbital Distance in Orange‐spotted Grouper,Epinephelus coioides (Hamilton, 1822)

The aim of this study was to investigate single nucleotide polymorphisms (SNPs) in genes that are presumed to control muscle growth and to determine their potential association with growth in a cultured population of orange‐spotted grouper, Epinephelus coioides. Seven genes, myogenic regulatory factor 4 (MRF4), Myf5, MSTN‐1, MSTN‐2, MyoD1, MyoD2, and myogenin, were selected for the investigation, covering approximately 26 kbp. First, the two clades for the genes MSTN (myostatin) and MyoD were confirmed in this species using Bayesian inference analysis of the phylogenetic relationships. Then, the seven genes were enriched by polymerase chain reaction and sequenced using an Ion Torrent Personal Genome Machine. A total of 586 SNPs were discovered. Linkage disequilibrium was decayed by 50% within 250 bp based on the combined data, which means that there was high resolution in the association mapping. A mixed linear model considering the population structure and kinship was used to detect the associations between genotypes and phenotypes. Only one site (KR269814.1:g.22T>G) in MSTN‐1 was found to be significantly associated with a measured trait, the interorbital distance (false discovery ratio < 0.05), and it explained 12.4% of the phenotype variation of this trait. This study provides insight on strategies for molecular marker‐assisted breeding in orange‐spotted grouper.     

The responsive expression of a caspase gene in Chinese shrimp Fenneropenaeus chinensis against pH stress

Caspases are a family of proteases, which play an important role in apoptosis. To evaluate the relationship between apoptosis and pH stress in crustaceans, a caspase gene (FcCasp) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length of FcCasp was 1329 bp with a 972 bp ORF, encoding a polypeptide of 323 amino acids with a calculated molecular weight and pI of 36.0 kDa and 6.27 respectively. The deduced amino acid sequence of FcCasp contained a potential active site (QACRG pentapeptide) conserved in most caspases and two profile hits (p20 and p10 domain profile). Comparison of amino acid sequences revealed that FcCasp had an overall similarity of 76–83% with other penaeid shrimp caspases. The amino acid sequence of recombinant FcCasp protein expressed in Escherichia coli was identified by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometer analysis. High‐level expression of FcCasp in six different tissues was detected by real‐time polymerase chain reaction after exposure to pH stress for 96 and 148 h. TUNEL analysis indicated that apoptosis began to appear in F. chinensis hepatopancreas exposed to extreme pH for 12 h. The amount of apoptosis seems positively correlated with the length of exposure to the pH stressor. The results suggested that FcCasp was involved in the response to environmental pH stress.     

暗纹东方鲀芳香化酶基因的结构及表达分析

性腺型芳香化酶基因是一种调节雌雄激素平衡的基因。为初步探究暗纹东方鲀性腺型芳香化酶基因在性腺发育和性别分化过程中的作用,通过RACE及荧光定量PCR技术,成功克隆出暗纹东方鲀性腺型芳香化酶基因的cDNA全长序列,共1786 bp,编码514个氨基酸,与红鳍东方鲀及星点东方鲀同源性最高。氨基酸序列分析显示,该基因编码的蛋白是一种稳定的亲水性蛋白,但不存在信号肽序列;性腺型芳香化酶含有38个磷酸化位点、4个N-糖基化位点、2个跨膜保守结构域;其氨基酸序列在哺乳动物和鱼类中均十分保守,且含有跨膜区、Ⅰ-螺旋区、Ozol′s肽区、芳香化酶特异性保守区和亚铁血红素结合区等功能保守区。表达分析显示,暗纹东方鲀性腺型芳香化酶基因主要在肌肉和卵巢中表达,其次在其他组织中也有少量表达,而且在幼鱼不同发育期卵巢中的表达量呈现出逐渐升高再降低的表达趋势,在精巢中则基本不表达。研究结果表明,性腺型芳香化酶基因很可能参与了暗纹东方鲀雌鱼性腺分化后卵巢的发育、雌性特征的维持和其他组织的发育等过程。     

EsWnt-4基因的表达分析

Wnt-4基因是Wnt基因家族重要的成员,在许多生物事件中都发挥着重要的作用。从中华绒螯蟹(Eriocheir sinensis)卵巢转录组数据库中筛选到EsWnt-4的cDNA序列1027 bp。RT-PCR检测发现EsWnt-4在神经组织、心脏和性腺中有表达,说明在中华绒螯蟹中EsWnt-4可能与多种组织细胞的活动有关。荧光定量检测发现,受精卵中EsWnt-4 mRNA的含量较成熟卵细胞和2、4、8细胞胚胎时期高,推测EsWnt-4基因有可能在受精卵中已经开始表达,而在之后的2、4、8细胞时期其表达又受到抑制。此外,EsWnt-4在16细胞期表达量再次升高,说明该时期可能有相关的生物学事件需要EsWnt-4基因的参与。在幼体阶段EsWnt-4在第二溞状幼体及第五溞状幼体阶段表达最高,说明EsWnt-4可能与幼体体节生成及变态发育有关。     

Molecular cloning and functional characterization of the fatty acid delta 6 desaturase (FAD6) gene in the sea cucumber Apostichopus japonicus

The sea cucumber (Apostichopus japonicus), an important echinodermata, had high value in nutrition and medicine for its rich collagen, sulphated polysaccharide, glycosides and polyunsaturated fatty acids (PUFA). The cDNA of the fatty acid desaturase gene in A. japonicus (AJFAD6) was cloned and was found to encode a desaturase with delta 6 FAD activity. Sequence analysis indicated that AJFAD6 included an open reading frame of 1392 bp, encoding 463 amino acids. AJFAD6 has all the conserved motifs found in other members of the FAD6 family, including an N‐terminal cytochrome b5 domain and three histidine‐rich regions. qRT‐PCR showed that AJFAD6 was expressed in all tissues tested during juvenile development and was mainly expressed in the respiratory tree at 150 days after adherence (150 days) and in the intestine at 100 days. Furthermore, AJFAD6 mRNA was also detected in the analysed adult tissues, with higher expression in the intestine and testis. Functional characterization of AJFAD6 in a recombinant yeast, Pichia pastoris, showed that AJFAD6 could catalyse exogenous linoleic acid (LA) and α‐linolenic acid (ALA) to produce γ‐linoleic acid (GLA) and stearidonic acid (STA), respectively, at conversion rates of 11.1% for LA to GLA and 3.4% for ALA to STA. Our results suggested that the biosynthetic pathway of PUFA existed in the sea cucumber, but endogenous production of eicosapentaenoic acid, arachidonic acid and docosahexaenoic acid from either LA or ALA precursor appeared to be limited.     

Molecular cloning and functional analysis of Photobacterium damselae subsp. piscicida haem receptor gene

A haem receptor gene from Photobacterium damselae subsp. piscicida (formerly known as Pasteurella piscicida) has been cloned, sequenced and analysed for its function. The gene, designated as pph, has an open reading frame consisting of 2154 bp, a predicted 718 amino acid residues and exists as a single copy. It is homologous with the haem receptors of Vibrio anguillarum hupA, V. cholerae hutA, V. mimicus mhuA and V. vulnificus hupA at 32.7, 32.7, 45.6 and 30.9%, respectively, and is highly conserved, consisting of a Phe-Arg-Ala-Pro sequence (FRAP), an iron transport related molecule (TonB) and a Asn-Pron-Asn-Leu sequence (NPNL), binding motifs associated with haem receptors. As a single gene knockout mutant P. damselae subsp. piscicida was able to bind haem in the absence of pph, suggesting that other receptors may be involved in its iron transport system. This study shows that the P. damselae subsp. piscicida pph belongs to the haem receptor family, is conserved and that its iron-binding system may involve more than one receptor.     

Enhanced muscle regeneration in freshwater prawn Macrobrachium rosenbergii achieved through in vivo silencing of the myostatin gene

Myostatin (MSTN) is an interesting negative growth‐regulating gene that has been well characterized in vertebrates but scantly described in invertebrates. The current study focuses on the downregulation of the MrMSTN gene and subsequently records any histological changes for giant freshwater prawn, Macrobrachium rosenbergii (Mr). In addition, the study also deals with the MrMSTN gene's influence on other growth‐related genes, which include myosin heavy chain, dystrophin‐dystroglycoprotein complex, tropomyosin, farnesoic acid o‐methyl transferase, arginine kinase, cyclophilin, and acyl CoA desaturase. The preliminary histological analysis following MrMSTN silencing favors muscle regeneration, which supports its functional role as a negative growth regulator and its significant effect on the expression of other growth‐related genes. Overall, our results show that the MrMSTN gene could therefore be a potential target for gene manipulation aimed at enhancing the growth and muscle development of M. rosenbergii, which could be beneficial in increasing the total mass production in the postlarva phase at the hatchery level.     

半滑舌鳎Vasa基因cDNA克隆及其在繁殖周期的表达

通过简并引物和RACE技术分离得到了半滑舌鳎Vasa基因的cDNA序列,其全长为2 602 bp,编码包含722个氨基酸残基的蛋白质。半滑舌鳎Vasa在N末端含有5个RGG重复和10个RG重复,具备DEAD-box家族特有的8个保守基序,与东方蓝旗鲔的同源性最高(91%)。利用RT-PCR技术研究了csVasa在性成熟半滑舌鳎中的表达模式。组织分布研究发现,Vasa在半滑舌鳎的卵巢和精巢中表达丰富,心脏中有微量表达;繁殖周期中,卵巢中Vasa的表达量在初级卵黄期和囊泡期要明显高于核周期和退化吸收期;精巢中,Vasa在精子细胞增殖期的表达量要显著高于精子成熟期和精母细胞增殖期。结果表明,Vasa可能在半滑舌鳎的配子发生中起重要作用。