马氏珠母贝载脂蛋白-2基因的克隆与表达分析

以RACE技术克隆获得马氏珠母贝载脂蛋白-2(Pm-ApoL2)基因cDNA全长序列,以传统萃取法提取黄色与白色闭壳肌个体的类胡萝卜素,以荧光定量技术检测Pm-ApoL2基因在各个组织中的表达量及黄与白闭壳肌个体的Pm-ApoL2表达量。结果表明,Pm-ApoL2基因cDNA序列全长1 328 bp,开放阅读框(ORF)长705 bp,编码234个氨基酸,5′非翻译区(5′UTR)长342 bp,3′UTR长281 bp。Pm-ApoL2在肝胰腺中表达量最高,其后依次是外套膜、鳃、闭壳肌,其中肝胰脏表达量显著大于其它3个组织(P0.05)。黄、白色闭壳肌个体之间的类胡萝卜素含量和Pm-ApoL2的表达量都存在显著性差异(P0.05),且二者的类胡萝卜素含量与Pm-ApoL2表达量呈显著正相关。Pm-ApoL2基因为马氏珠母贝类胡萝卜素代谢候选基因。     

马氏珠母贝B型清道夫受体PmSR-B基因克隆与表达性分析

采用c DNA末端快速扩增技术克隆获得马氏珠母贝清道夫受体基因c DNA全长序列(Pm SR-B);利用荧光定量技术检测Pm SR-B基因在各个组织中的表达量。结果表明,Pm SR-B基因c DNA序列全长1 857 bp,开放阅读框(ORF)长1 443 bp,编码480个氨基酸,5′非翻译区(5′UTR)长89 bp,3′UTR长325 bp。预测其分子质量为54.77 ku,等电点为7.94,脂溶性系数92.94,总平均亲水性-0.120,属于疏水性蛋白;不稳定指数26.37,属于稳定蛋白。氨基酸序列同源比对结果,Pm SR-B氨基酸序列与其他物种具有一定的保守性,与华贵类栉孔扇贝(Mimachlamys nobilis)SR-B的序列的相似度高达47%。荧光定量PCR检测结果,Pm SR-B在肝胰腺中表达量最高,其后依次是鳃、闭壳肌、中央膜,各组织的表达量差异具统计学意义(P0.05)。     

Molecular Characterization and Expression and DNA Methylation Analyses of a Galectin-Related Protein Gene from Cynoglossus semilaevis

Galectins,a family of ?-galactoside-binding proteins,participate in both innate immunity and adaptive immunity.This study identified one novel galectin-related protein from half-smooth tongue sole Cynoglossus semilaevis,which was designated as Cs GRP.The full-length c DNA of its encoding gene was 785 bp in length with a 528 bp open reading frame encoding a putative protein of 176 amino acids.The deduced Cs GRP contained a putative 131-aa galactoside-binding lectin domain and 3 critical residues responsible for carbohydrate binding(R~(93),W~(109) and R~(114)).Genomic structural analysis revealed that Cs GRP consisted of five exons and four introns.Cs GRP showed 68% similarity with Poecilia latipinna GRP and 67% similarity with Stegastes partitus GRP.Cs GRP showed the highest expression level in liver,although its expression was detected in all tested tissues.When challenged with Vibrio harveyi,the expression of Cs GRP was significantly down-regulated in liver(P 0.05).In addition,we found that in spleen and kidney of C.semilaevis,the Cp G island of Cs GRP showed significantly higher(P 0.05) methylation level in disease-resistant family of C.semilaevis(DR-Cs) than in disease-susceptible ones(DS-Cs).Our results suggested that Cs GRP may play important roles in the immune response of C.semilaevis.Moreover,DNA methylation patterns provided valuable data for understanding the relationship between epigenetic regulation and immunity,which would assist the animal genetic research and improve the animal breeding in the future.     

Molecular Cloning,Expression and Characterization of Peroxisome Proliferators-Activated Receptors Gamma in the Sea Urchin(Strongylocentrotus intermedius)

Peroxisome proliferators-activated receptor gamma(PPARγ) plays important regulatory roles in adipocyte differentiation. In this study, we cloned the full-length sequence of the PPARγ gene and analyzed its expression profile in different developmental stages and tissues of Strongylocentrotus intermedius. The full-length cDNA of PPARγ contains 2286 base pairs(bp) with a putative open reading frame of 1755 bp, and the gene encodes encoding a polypeptide of 584 amino acid residues. The predicted molecular mass of the protein is 67.27 kDa, and its theoretical isoelectric point(pI) is 10.07. The protein contains conserved motifs, including an RRM(RNA recognition motif) domain. PPARγ expression with the highest level was observed in the gonad, and the lowest was observed in the tube feet of S. intermedius. Time-course expression measurements at different developmental stages showed that the highest expression level of PPARγ is in the eggs and its weakest expression level is in the 32-cells stage. Knock-down of PPARγ by specific siRNA revealed that UCP2 expression is significantly decreased in the gonads and intestines 48 h post-transfection, indicating that the UCP2 is a downstream target gene of PPARγ.This finding suggests that PPARγ and UCP2 have positive regulatory effects in regulating adipocyte differentiation. Changes in fatty acid levels in the gonads before and after PPARγ interference were assessed, and decreased C18:2(trans, n-6) and C20:3(n-6) levels were observed 48 h after siRNA transfection. The results showed the function of PPARγ in fatty acid anabolism, The data are helpful to improve the current understanding of the fatty acid synthesis pathways and regulatory mechanisms in S. intermedius. They also provide an experimental basis for improving fatty acid synthesis in sea urchins, which is important for cultivating sea urchins with high nutritional value.     

Molecular Characterization and Expression Analysis of SKIV Infection of Interferon-Induced Protein with Tetratricopeptide Repeats 1(IFIT1) in Epinephelus lanceolatus

Interferon-induced protein with tetratricopeptide repeats 1(IFIT1), also known as interferon-induced protein 56(IFI56) or Interferon-stimulated protein 56(ISG56), was originally identified as a protein induced upon treatment with interferon and inhibited by viral replication and translational initiation. In this study, Epinephelus lanceolatus IFIT1(ELIFIT1) gene was cloned for the first time. The complete cDNA of El IFIT1 gene includes 2921 nucleotides, and encodes a 437-amino acid(AA) protein. The putative ELIFIT1 protein has 9 TRP domains and is highly similar with IFIT1 proteins in other teleosts. In healthy fish, ELIFIT1 gene was highly expressed in the blood, which indicate its specific function in the peripheral immune system. Its expression was also observed in various immunity-related tissues including spleen, intestine, and kidney, Inducted with spotted knifejaw iridovirus(SKIV), ELIFIT1 gene expression was upregulated in the spleen, kidney, and liver 24 h after induction and reached its peak at 72 h, indicating that ELIFIT1 may play an important role in antivirus. These findings contribute to the understanding of the antiviral regulation of ELIFIT1 gene in teleost.     

Characterization and Expression Analysis of a Complement Component Gene in Sea Cucumber (Apostichopus japonicus)

The complement system plays a crucial role in the innate immune system of animals. It can be activated by distinct yet overlapping classical, alternative and lectin pathways. In the alternative pathway, complement factor B(Bf) serves as the catalytic subunit of complement component 3(C3) convertase, which plays the central role among three activation pathways. In this study, the Bf gene in sea cucumber(Apostichopus japonicus), termed Aj Bf, was obtained by rapid amplification of c DNA ends(RACE). The full-length c DNA of Aj Bf was 3231 bp in length barring the poly(A) tail. It contained an open reading frame(ORF) of 2742 bp encoding 913 amino acids, a 105 bp 5'-UTR(5'-terminal untranslated region) and a 384 bp 3'-UTR. Aj Bf was a mosaic protein with six CCP(complement control protein) domains, a VWA(von Willebrand factor A) domain, and a serine protease domain. The deduced molecular weight of Aj Bf protein was 101 k Da. Quantitative real time PCR(q RT-PCR) analysis indicated that the expression level of Aj Bf in A. japonicus was obviously higher at larval stage than that at embryonic stage. Expression detection in different tissues showed that Aj Bf expressed higher in coelomocytes than in other four tissues. In addation, Aj Bf expression in different tissues was induced significantly after LPS or Poly I:C challenge. These results indicated that Aj Bf plays an important role in immune responses to pathogen infection.     

Cloning,Expression and Characterization of a Lipase Gene from Marine Bacterium Pseudoalteromonas lipolytica SCSIO 04301

Absract A lipase gene, lip1233, isolated from Pseudoalteromonas lipolytica SCSIO 04301, was cloned and expressed in E. coli. The enzyme comprised 810 amino acid residues with a deduced molecular weight of 80 k Da. Lip1233 was grouped into the lipase family X because it contained a highly conserved motif GHSLG. The recombinant enzyme was purified with Ni-NTA affinity chromatography. The optimal temperature and p H value of Lip1233 were 45℃ and 8.0, respectively. It retained more than 70% of original activity after being incubated in p H ranging from 6.0 to 9.5 for 30 min. It was stable when the temperature was below 45℃, but was unstable when the temperature was above 55℃. Most metal ions tested had no significant effect on the activity of Lip1233. Lip1233 remained more than original activity in some organic solvents at the concentration of 30%(v/v). It retained more than 30% activity after incubated in pure organic solvents for 12 h, while in hexane the activity was nearly 100%. Additionally, Lip1233 exhibited typical halotolerant characteristic as it was active under 4M Na Cl. Lip1233 powder could catalyze efficiently the synthesis of fructose esters in hexane at 40℃. These characteristics demonstrated that Lip1233 is applicable to elaborate food processing and organic synthesis.     

Establishment,Characterization and Expression Pattern of a Spleen Cell Line,SMSP, Derived from Turbot(Scophthalmus maximus)

A turbot(Scophthalmus maximus) cell line named SMSP was obtained from the spleen. The origin of the cells was identified by morphology, chromosome number and COI gene. The optimal basic medium, serum concentration and growth temperature of the cells were detected. SMSP cell line is mainly composed of fibroblast-like cells. Most of the SMSP cells contained 44 chromosomes, and the sequence of COI gene confirmed that the cells were originated from turbot. The optimal culture conditions were 24℃,DME...     

北斗三号新信号B2a和B2（B2a+b）质量分析及星座几何性能评估

在最新的能接收到北斗三号新信号B2a及B2（B2a+b）的MGEX测站数据基础上，分析BDS-3信号的载噪比、多路径及卫星的三维位置精度因子（PDOP）。结果表明，与GPS相比，BDS的载噪比B2（B2a+b）>B2a≈L5>B3≈B2b>B1≈L1>L2，多路径误差B2(B2a+b)相似文献