Characterization of a RacGTPase up-regulated in the large yellow croaker Pseudosciaena crocea immunity

The Rac proteins are members of the Rho family of small G proteins and are implicated in the regulation of several pathways, including those leading to cytoskeleton reorganization, gene expression, cell proliferation, cell adhesion and cell migration and survival. In this investigation, a Rac gene (named as LycRac gene) was obtained from the large yellow croaker and it was expressed in Escherichia coli and purified. Subsequently the specific antibody was raised using the purified fusion protein (GST-LycRac). Moreover, the GTP-binding assay showed that the LycRac protein had GTP-binding activity. The LycRac gene was ubiquitously transcribed and expressed in 9 tissues. Quantitative real-time RT-PCR and Western blot analysis revealed the highest expression in gill and the weakest expression in spleen. Time-course analysis revealed that LycRac expression was obviously up-regulated in blood, spleen and liver after immunization with polyinosinic polycytidynic acid (poly I:C), formalin-inactive Gram-negative bacterium Vibrio parahemolyticus and bacterial lipopolysaccharides (LPS). These results suggested that LycRac protein might play an important role in the immune response against microorganisms in large yellow croaker.     

Isolation and characterization of 11 microsatellite markers for the large yellow croaker, Pseudosciaena crocea

A new set of 11 microsatellite markers was isolated and characterized in the large yellow croaker (Pseudosciaena crocea Richardson) from a CA-enriched genomic library. Of 35 microsatellite markers screened, 11 microsatellite markers are highly polymorphic in one hatchery stock which contained 58 individuals. The high genetic variability was represented mainly by the number of alleles, which ranged from 7 to 16, and the observed heterozygosity, which ranged from 0.43 to 0.79. These newly isolated markers increase the available molecular resources that can be used to analyze genetic diversity and population structure of large yellow croaker.     

Cloning and molecular characterization of lycC1INH genes in large yellow croaker (Pseudosciaena crocea)

Complement component 1 inhibitor (C1INH) is a crucial protein in controlling activation of many plasma mediator pathways and can directly interact with Gram negative bacteria. The full-length cDNA of lycC1INH gene was identified from the large yellow croaker. It is of 2046 nucleotides (nt) encoding a protein of 599 amino acids, with a 5′-untranslated region of 99 nt and a 3′-untranslated region of 147 nt including the poly (A) tail. The deduced protein contains a C-terminal serpin (serine protease inhibitor) domain, and two N-terminus immunoglobulin domains without significant homology to other species. Western blot analysis of the protein expression showed that the expression of lycC1INH was obviously up-regulated in liver, spleen and head kidney of the fish challenged by attenuated live Vibrio anguillarum strain. This indicated that lycC1INH might be involved in the immune response of large yellow croaker to bacterial challenge.     

Molecular characterization and bioactivity of a CXCL13 chemokine in large yellow croaker Pseudosciaena crocea

A CXCL13-like chemokine cDNA was isolated from large yellow croaker (Pseudosciaena crocea) by expressed sequence tag (EST) analysis (LycCXCL13). The full-length cDNA of LycCXCL13 is 796 nucleotides (nt) encoding a protein of 97 amino acids (aa), with a putative molecular weight of 10.7 kDa. The deduced LycCXCL13 contains a 24-aa signal peptide and a 73-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines (C²⁵, C²⁷, C⁵² and C⁶⁸). It shares 35, 36 and 39% aa sequence identities to green puffer CXCL13-like, Atlantic salmon CXCL13 and Japanese flounder CXCL13 chemokines, and 24–29% identities to CXCL13 chemokines in mammals, respectively. Phylogenetic analysis showed that LycCXCL13 is more closely related to the CXCL13 subgroup than to any other CXC chemokine subgroups. LycCXCL13 gene was constitutively expressed in all tissues examined, except for intestine. Upon induction with poly(I:C) or inactivated trivalent bacterial vaccine, LycCXCL13 gene expression was significantly up-regulated in spleen, head kidney, heart and gills at 24 h post-injection. Real-time PCR results showed that LycCXCL13 gene expression reached peak level in spleen and head kidney at 12 h after induction by poly(I:C), while its expression increased to the highest level in head kidney at 24 h or in spleen at 48 h by bacterial vaccine. Recombinant LycCXCL13 protein produced in E. coli BL21 exhibited obvious chemotaxis to the peripheral blood leucocytes (PBLs) from large yellow croaker. These results suggest that LycCXCL13 may be involved in inflammatory responses as well as homeostatic processes in large yellow croaker.     

Effects of starvation on lipid accumulation and antioxidant response in the right and left lobes of liver in large yellow croaker Pseudosciaena crocea

The present study aimed to determine the effects of starvation on lipid content and antioxidant responses in the right and left lobes of liver in large yellow croaker. Fish were divided into three groups: the control fish fed normally and the fish starved for 4 and 12 days. The set of biomarkers were determined, including crude lipid and MDA contents, and mRNA levels and activities of copper and zinc superoxide dismutase (Cu/Zn-SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR). Starvation for 12 days decreased lipid content and increased MDA content and mRNA levels and activities of antioxidant enzyme genes tested in both lobes of liver. No significant difference in these biomarkers between both lobes of liver was observed in fish starved for 12 days. However, there were significant differences between both lobes of liver in lipid and MDA contents, activities of CAT and GR, and expression levels of Cu/Zn-SOD and GR in fish starved for 4 days. These observed differences between starved and fed fish and between both lobes of liver could be important biomarkers that contributed in separating starved from fed fish and short-term starved from long-term starved fish, respectively. Our study emphasized the same lobe of the liver should be sampled when evaluating biomarkers during starvation in fish.     

Eleven new highly polymorphic microsatellite loci in the yellow croaker,Pseudosciaena crocea

We developed 11 new microsatellite markers in Pseudosciaena crocea by screening an enriched genomic library using nonradioactive polymerase chain reaction (PCR) techniques. All loci were found to be polymorphic with an average of 14.9 alleles per locus (range four to 30). The mean observed and expected heterozygosities were 0.86 (range 0.57–1.00) and 0.90 (range 0.62–0.98), respectively. Four loci showed significant Hardy–Weinberg disequilibrium. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for population genetic studies of P. crocea.     

Molecular cloning and functional characterization of a RabGTPase in large yellow croaker (Pseudosciaena crocea)

The molecular mechanisms of the immune system against pathogens in large yellow croaker (Pseudosciaena crocea) are not well known, despite its economic importance as an aquaculture species. In this investigation, a Rab gene (named as LycRab gene) was obtained from this fish, which exhibited high homology with Rab8 of other species. It was expressed in Escherichia coli, and the specific antibody was raised using the purified fusion protein (GST-LycRab). The LycRab protein, containing characteristic signatures of Rab proteins with 5 GTP-binding domains, had GTP-binding activity. The LycRab gene was ubiquitously expressed in all analyzed tissues as revealed by Western blot, although expression levels varied from tissue to tissue. Real-time PCR revealed that the LycRab gene was up-regulated after immunization with poly I:C, formalin-inactive Gram-negative bacterium Vibrio parahaemolyticus or bacterial lipopolysaccharides (LPS), suggesting that LycRab protein might play an important role in large yellow croaker defense against pathogens infection. This discovery might contribute better understanding to the molecular events involved in fish immune responses.     

大黄鱼对几种饲料蛋白原料消化率的研究

以白鱼粉为主要蛋白源制成参考饲料,以0.1%的Cr2O3为外源性指示剂,按参考饲料和实验原料70%∶30%的比例配制成实验饲料,测定了大黄鱼（Pseudosciaena crocea）对白鱼粉、红鱼粉、肉骨粉、豆粕、花生粕、菜籽粕和棉籽粕的表观消化率。实验大黄鱼（平均初始体重15.0±1.6g）养殖于海水浮式网箱（1.5m×1.5m×2.0m）中,分别以各实验饲料投喂实验鱼5周,然后采用挤压法收集粪便。实验结果表明各原料表观干物质消化率在43.6%至70.0%之间,能量消化率在42.9%到82.6%的范围内。实验原料的蛋白质表观消化率在70.7%到92.4%之间,其中白鱼粉和红鱼粉的蛋白消化率最高（分别为92.4%和89.3%）,而棉籽粕则最低（70.7%）。脂肪的表观消化率在61.3%到90.5%之间,其中棉籽粕最低（61.3%）。磷的表观消化率相对较低,仅白鱼粉和红鱼粉在53.2%以上,其余皆低于37.5%。总之,大黄鱼对这几种原料的表观消化率存在较大差异,但蛋白消化率均较高,因此,可作为大黄鱼的饲料蛋白源在实际饲料配制中使用。     

大黄鱼耐低温性状相关微卫星标记的筛选

鱼类的耐低温性状是一种重要的经济性状。为了初步分析大黄鱼的耐低温性状, 文章采用15对荧光微卫星标记, 以SSR-PCR方法对大黄鱼低温耐受组和正常对照组F₁代共40个个体进行了耐低温性状遗传差异分析。结果显示, 标记LYC0002在两组样品中共扩增出5个等位基因(片段大小分别为112 bp、110 bp、108 bp、106 bp和104 bp), 其中LYC00021_{12 bp}等位基因在低温耐受组的出现频率达60%, 而在正常对照组中的频率为零, 表明该等位基因对大黄鱼的温度敏感特性有较明显的偏好性, 可能与某种耐低温基因存在一定的连锁关系。此外, 对LYC0002_{106 bp、}LYC0002_{108 bp}、LYC0002_{110 bp} 和 LYC0002_{112 bp} 4个等位基因分别进行了回收、克隆及测序。序列比对结果显示, LYC0002_{112 bp}等位基因含有10个(CA)重复单元, 而其他3个等位基因依次缺失1个(CA)重复单元, 说明LYC0002在本研究样本中的突变方式为微卫星逐步突变模型(Stepwise mutation model, SMM)。     

Isolation and characterization of six microsatellite markers in the large yellow croaker (Pseudosciaena crocea Richardson)

Six polymorphic microsatellite markers were isolated and characterized using an enriched library technique in the large yellow croaker (Pseudosciaena crocea Richardson, 1864), a commercially important marine fish in China. They showed PIC (polymorphism information content) ranging from 0.064 to 0.885 (average of 0.580) and allele numbers ranging from two to 13 (average of 7.5), which were useful for the studies on population genetics and selective breeding of the large yellow croaker.