Cloning and characterization of phospholipase D from olive flounder (Paralichthys olivaceus)

The phospholipase D1 (PLD1) cDNA, designated PoPLD, encoding a predicted protein of 1053 amino acids in olive flounder (Paralichthys olivaceus) has been cloned. The deduced amino acid sequence shares high identity with that of PLD1s and PLD2 in human, rat and mouse. The phylogenic analysis and sequence comparison of PoPLD with other PLD isozymes were found to be closely related to the PLD1 isozyme in primary structure. The tissue expression analysis of PoPLD showed that the mRNA of PoPLD was predominantly expressed in the brain, gullet, muscle, stomach, head kidney, pyloric caeca, intestine and gill. The expression of the PoPLD gene was examined in various tissues of flounder by RT-PCR following stimulation with LPS and compared also with that of the inflammatory cytokines IL-1beta and IL-8 in various tissues of the stimulated flounder. This provides indirect evidence that PLD1 might have a relevant role in immune responses against pathogens and in inflammation. In addition, the recombinant protein of PoPLD (GFP-PoPLD), which demonstrated a phosphatidylcholine (PC)-hydrolyzing activity, was partially localized as a distinct ring-shaped form surrounding the rim of the nucleus in EPC cells. Together, our results suggest that PoPLD is similar to the mammalian PLD1 isoform, is generally widespread within olive flounder tissue, might have a relevant role in the fish immune system against pathogens and specifically may be localized in the subcellular membranes of the nuclear rim in EPC cells.     

Gene expression of leucocytes in vaccinated Japanese flounder (Paralichthys olivaceus) during the course of experimental infection with Edwardsiella tarda

In this paper, we focused on the detection of differentially expressed genes in peripheral blood leucocytes (PBL) during the course of Edwardsiella tarda infection in vaccinated and non-vaccinated Japanese flounder (Paralichthys olivaceus). cDNA microarray analysis was performed to compare the gene expression patterns of the PBL between the vaccinated and non-vaccinated fish in response to E. tarda inoculation. Fish were vaccinated twice, at a two-week interval and experimentally challenged with E. tarda two weeks after the second vaccination. Among the 1187 analyzed genes, 42 genes were up-regulated during the course of infection either in vaccinated or non-vaccinated fish. These genes included immune-related genes, such as MMP-9, MMP-13, CXC chemokine, CD20 receptor and hepcidin. Some immune-related genes were down-regulated after the E. tarda challenge, i.e. interferon inducible Mx protein, MHC class II-associated invariant chain, MHC class II alpha and MHC class II beta encoding genes, immunoglobulin light chain precursor, immunoglobulin light chain and IgM. These responses are thought to be a common reaction of Japanese flounder PBL in the course of edwardsiellosis, irrespective of immunized condition. Ten genes were significantly up-regulated only in vaccinated fish, and 11 genes were significantly up-regulated only in non-vaccinated fish. These genes may have a correlation with the efficacy of vaccination, although we have no evidence to link the different gene expression patterns and the efficacy of vaccination at present.     

微卫星标记对牙鲆有丝分裂雌核发育家系的亲子鉴定

利用18个微卫星标记,对6个家系的26尾有丝分裂雌核发育牙鲆进行亲子鉴定,PCR扩增产物经8%非变性聚丙烯酰胺凝胶电泳检测,结果表明:1个座位在母本中表现为相同的基因型,视为单态座位,其他17个座位为多态;多态座位在亲子鉴定中的累计排除概率和累计个体识别概率分别为0.9985、0.9999;根据被测个体在17个微卫星座位的基因型,最后确认26尾子代的母本,其中7尾子代在某些座位表现出与其母本不完全匹配的基因型。利用微卫星标记可确定雌核发育后代的亲子关系,从而构建牙鲆雌核发育家系系谱,对牙鲆雌核发育的深入研究具有重要意义。     

微卫星评价牙鲆雌核发育二倍体纯合性

采用8个微卫星座位分别对牙鲆减数雌核发育二倍体家系和卵裂雌核发育二倍体家系的纯合性进行检验。卵裂雌核发育二倍体在所有检测座位全部纯合。减数雌核发育二倍体在部分座位发生纯合,但未发现在所有座位全部纯合的个体,在Poli9TUF、Poli9-8TUF、Poli11TUF、Poli13TUF、Poli23TUF、Poli30TUF、Poli123TUF和Poli130TUF座位,杂合子比例分别为1·0000、1·0000、0·1944、0·9459、0·8611、1·0000、0·7778和0·8000,平均杂合子比例为0·8224。由此表明,牙鲆除了Poli11TUF外,在其余7个座位均具有很高的重组率。研究结果显示,牙鲆卵裂雌核发育二倍体一代即可形成纯合子;而减数雌核发育二倍体由于具有较高的重组率,使其与母本的遗传同质性较高。     

龙眼体胚发生过程中DCL基因的分子特性及表达分析

从龙眼转录组unigene序列筛选获得龙眼DCL基因(命名为DlDCL)全长序列,并结合生物信息学及实时荧光定量等方法,对龙眼DCL基因进行研究,以明确DlDCLs在龙眼体胚、不同生长组织部位中的表达规律及其对激素和光质应答反应,为进一步研究龙眼体胚过程中DlDCLs基因的调控研究奠定基础。结果表明:(1)龙眼转录组数据存在DlDCL1、DlDCL2、DlDCL3和DlDCL4四个DCL家族成员,且基于Unigene的FPKM值发现不同基因在体胚发生阶段具有差异表达。(2)生物信息学分析发现,DlDCLs成员间基本理化性质较为类似,均为亲水性不稳定蛋白、不含信号肽、可进行跨膜运动,但也存在一定差异,如DlDCL2为碱性蛋白,而其他3个成员为酸性蛋白;亚细胞定位预测显示,DlDCLs均定位于细胞核中,但DlDCL2也存在定位于叶绿体中;对DCL蛋白结构域预测显示,DCL是高度保守的蛋白。(3)系统进化树分析显示,不同物种的DCL分为4个分支,同源的DCL蛋白都聚为一类,且DlDCLs与柑橘DCL亲缘关系更为接近。(4)实时荧光定量PCR分析表明,DlDCLs在龙眼非胚性愈伤组织和体胚发生过程中的表达模式差异较大,但DlDCLs在愈伤组织阶段均有较高的表达量,推测DlDCLs在体胚发生过程可能具有功能的独立和协作。DlDCL1和DlDCL2在叶片、花器官等组织部位中的相对表达量较高,暗示DlDCL1和DlDCL2可能参与到光合作用和花器官的发育;DlDCLs还受2,4-D、MeJA、SA激素和光质诱导,表明DlDCLs可能参与激素和光质调控。     

Molecular cloning, expression, and functional analysis of caspase-10 from Japanese flounder Paralichthys olivaceus

We isolated and sequenced caspase-10 cDNA and gene from Japanese flounder, Paralichthys olivaceus. The Japanese flounder (JF)-caspase-10 cDNA consisted of 2282 bp and encoded 495 amino acid residues. The characteristic death effector domains (DEDs) of caspases were observed in JF-caspase-10 as well as the three aspartic acid residues (D-186, -382 and -392), which are potential cleavage sites for the large and small subunit structures. The amino acid residue (His-325) and pentapeptide (QACQG), which are involved in catalytic activity, were absolutely conserved in Japanese flounder-caspase-10. JF-caspase-10 gene has a length of 6.6 kb and consists of 11 exons and 10 introns similar to that of human. The strong expression of JF-caspase-10 mRNA was detected in the gills, peripheral blood leukocytes, spleen and posterior kidney, while the weak expression was observed in the head kidney, heart, intestine, skin and stomach. The over-expression analysis of JF-caspase-10 in Japanese flounder cell line HINAE was shown to induce apoptosis 24h post-transfection using TUNEL assay.     

Effects of long-term starvation on hepatocyte ultrastructure of olive flounder Paralichthys olivaceus

The effects of nutritional conditions on alterations in condition factor, liver-somatic index, and hepatocyte ultrastructure in the olive flounder Paralichthys olivaceus were examined. Twelve weeks of starvation significantly decreased the condition factor and the liver-somatic index in the olive flounder. Hepatocytes underwent marked ultrastructural changes in response to 12 weeks of starvation. Compared to those of the initial control and fed group, the prominent features characterizing the hepatocytes of the starved group were: reduction in cell and nucleus size; apparent loss of nucleoli; condensation of chromatin; loss of stored glycogen; reduction of endoplasmic reticulum profile; increase in the number of electron-dense bodies containing large amounts of iron; and increased mitochondrial size. Results suggest that the histological changes caused by ultrastructural alterations in the hepatocytes can be used as alternative indicators to identify starvation in cultured P. olivaceus.     

Sequences of 596 cDNA Clones (565,977 bp) of Japanese Flounder (Paralichthys olivaceus) Leukocytes Infected with Hirame Rhabdovirus

We have partially sequenced 785 sequences of 596 independent complementary DNA clones isolated from a cDNA library of Japanese flounder leukocytes infected with hirame rhabdovirus. These sequences consist of a total of 565,977 base pairs. The average size of the sequenced lengths was 721 bp. Of 596 clones, 386 (64.8%) were identified as previously reported genes by the BLASTN and BLASTX programs. About 30% of the identified clones could be recognized by only the BLASTX program. A total of 251 distinct genes were identified, and 181 of these genes are the first such genes reported from the teleostei. Approximately 27% of the identified Japanese flounder genes appear to be associated with cell division, cell structure or motility, and basic energy metabolism, 29% with gene or protein expression, and 17% with cell signaling, cell communication, and cell or organism defense. The most frequently identified expressed sequence tags of leukocytes of Japanese flounder were gelatinase b and ribosomal protein L23, which both had 1.34% prevalence. Received February 12, 1999; accepted April 13, 1999     

不同代次牛成肌细胞的诱导分化及相关基因的表达分析

利用初生荷斯坦牛的成肌细胞,在不同代次以含体积分数为2%马血清的DMEM进行诱导分化,在之后0、2、4、6、8、10 d观察细胞的形态变化并收集细胞提取总RNA,检测成肌相关基因的表达情况,为进一步研究牛肌肉发生过程及相关基因的表达调控提供依据。同时利用实时荧光定量PCR分别检测肌性相关基因MyoD(生肌决定因子)、MyoG(肌细胞生成素)以及非肌性相关基因A-FABP(脂肪细胞型脂肪酸结合蛋白)表达水平的变化。研究表明：①各代细胞在诱导培养4 d后开始有肌管形成,其后越来越多,8 d时达高峰。②成肌细胞向成熟肌细胞分化过程中,MyoD、MyoG、A-FABP基因都呈先上升然后下降的趋势。③高代次成肌细胞比低代次细胞增殖慢,而且在诱导分化后,肌管的数目明显变少; MyoD、MyoG、A-FABP随着代次的升高而下降。故推断MyoD、MyoG以及A-FABP基因会在成肌分化开始后的不同阶段被激活,从而发挥不同的调控作用,而且随着传代次数的增加成肌细胞的分化能力减弱。     

Cloning, Expression and Characterization of a Thiolase Gene from Clostridium pasteurianum

A thl gene encoding the thiolase (EC 2.3.1.9) of Clostridium pasteurianum was cloned by thermal asymmetric interlaced (TAIL) PCR. It consists of 1179 bp with 36.8% GC content and encodes 392 amino acids with a deduced molecular mass of 40,954 Da and shows 77% identity and 88% similarity to that of Clostridium tetani E88 and should be classified as a biosynthetic thiolase with three conserved residues Cys89, Cys382 and His352. The gene was over-expressed in Escherichia coli and the thiolase was purified with Ni-NTA agarose column to homogeneity. The K _m of this thiolase for acetoacetyl-CoA is 0.13 mM with 0.06 mM CoASH at pH 8.2, 25°C and a V _max value of 46 μmol min⁻¹ mg⁻¹.     

The development of the lymphoid organs of flounder, Paralichthys olivaceus, from hatching to 13 months

The growth of the lymphoid organs, such as head kidney, spleen and thymus were studied in flounder, Paralichthys olivaceus Temminck & Schlegel, from hatching to 13 months of age. Except for the thymus, all organs grew as the fish grew. By 2 months of age the lymphoid organs attained their maximum relative weight. The organ weight showed a closer correlation to body weight than they did to age. The total number of leucocytes in the lymphoid organs increased with age, but the number per milligram of lymphoid organ remained constant. A micro and ultrastructural study of the lymphoid organs showed that the full development of the lymphoid organs was not achieved until the juvenile stage. The spleen and head kidney had mixed populations of "red" and "white" cells. The head kidney was more lymphoid than the spleen. The thymus involuted quickly during the first 6 months. The blood components had no obvious relationship with age or season during the period studied.     

Isolation and characterization of new microsatellite markers from the Japanese flounder (Paralichthys olivaceus)

The isolation and characterization of eight polymorphic microsatellite loci from a Japanese flounder partial genomic library are reported. The eight markers isolated in this study were highly polymorphic and their positions on the linkage genome map of the Japanese flounder were determined. Therefore, they are useful for ecological studies of wild populations. These markers are more effective than other markers with no information of chromosomal locations.     

Effects of Cold Shock on Microtubule Organization and Cell Cycle in Gynogenetically Activated Eggs of Olive Flounder (Paralichthys olivaceus)

Cytological changes and subsequent mitotic processes were studied in gynogenetically activated eggs of olive flounder subjected to cold-shock treatment using indirect immunofluorescence staining of isolated blastodisks. Obvious differences between controls and treated eggs were detected during early cell division. The developmental process of haploid control was similar to that of the diploid control except several minutes delayed. Spindles disassembled by the cold-shock treatment regenerated soon after treatment, resulting in the occurrence of the first mitosis. The immature daughter centriole was easily depolymerized by cold-shock treatment, leading to the formation of the bipolar spindle in the first cell cycle and the formation of the monopolar spindle in the second cell cycle, resulting in chromosome set doubling. Some two-cell stage eggs had a monopolar spindle in one blastomere and a bipolar spindle in another during the second mitosis. These eggs had a high potency developing into haploid-diploid mosaics. To the best of our knowledge, this study is the first to clarify the mechanism of chromosome set doubling in marine fishes and provides a preliminary cytological basis for developing a reliable and efficient protocol for mitotic gynogenesis induction by cold-shock treatment in olive flounder.     

Molecular Characterization and Expression Analysis of ATP-Gated P2X7 Receptor Involved in Japanese Flounder (Paralichthys olivaceus) Innate Immune Response

ATP-gated P2X7 receptor (P2RX7) channel is a key component for purinergic signaling and plays important roles in the innate immune response in mammals. However, the expression, molecular properties and immune significances of P2RX7 in lower vertebrates are still very limited. Here we identified and characterized a novel bony fish P2RX7 homologue cDNA, termed poP2RX7, in Japanese flounder (Paralichthys olivaceus). PoP2RX7 protein shares about 60–88% sequence similarity and 45–78% sequence identity with known vertebrate P2RX7 proteins. Phylogenetic analysis placed poP2RX7 and other P2RX7 proteins within their own cluster apart from other P2RX members. While the functional poP2RX7 channel shares structural features in common with known P2RX7 homologs, electrophysiological studies revealed that BzATP, the more potent agonist for known mammalian and fish P2RX7s, shows similar potency to ATP in poP2RX7 activation. poP2RX7 mRNA constitutively expressed in all examined tissues from unstimulated healthy Japanese flounder with dominant expression in hepatopancreas and the lowest expression in head kidney, trunk kidney, spleen and gill. poP2RX7 mRNA expression, however, was significantly induced in Japanese flounder head kidney primary cells by Poly(I:C) and bacterial endotoxin LPS stimulations. In vivo experiments further revealed that poP2RX7 gene expression was substantially up-regulated by immune challenge with infectious bacteria Edwardsiella tarda and Vibrio anguillarum. Moreover, activation of poP2RX7 results in an increased gene expression of multifunctional cytokines IL-1β and IL-6 in the head kidney primary cells. Collectively, we identified and characterized a novel fish P2RX7 homolog which is engaged in Japanese flounder innate immune response probably through modulation of pro-inflammatory cytokines expression.     

麻竹同源异型盒基因DlKNOX的克隆及表达分析

为探讨同源异型盒(KNOX)基因在麻竹(Dendrocalamus latiflorus)茎秆发育中的作用,采用RT-PCR和RACE技术,从其幼茎中克隆了1个KNOX同源基因,命名为Dl KNOX,其c DNA序列全长为1511 bp,包含5′UTR 196 bp、3′UTR 238 bp和编码区1077 bp。该基因编码含358氨基酸的蛋白,具有KNOX1、KNOX2、ELK和Homeobox KN等4个保守结构域,符合KNOX家族的特征,属于I类蛋白。生物信息学分析表明,该基因编码的蛋白与水稻OSH1的一致性最高(86%)。组织表达特异性分析表明,Dl KNOX在节部的表达丰度最高,其次为幼茎,根中最低。Dl KNOX基因在大肠杆菌(Escherichia coli)中经诱导表达,获得1条分子量约为82 k Da的重组蛋白,与预期的重组蛋白分子量一致(包含了MBP标签蛋白42.5 k Da和Dl KNOX蛋白39.5 k Da)。该基因在大肠杆菌中的最适表达条件为28℃,0.3 mmol L–1 IPTG诱导2 h。这为进一步研究Dl KNOX在麻竹茎秆发育中的功能奠定了基础。     

龙眼体细胞胚胎发生过程中DlWUS的克隆与表达分析

以龙眼‘红核子’LC2悬浮细胞系诱导的胚性愈伤组织为基本材料,按照龙眼体细胞胚胎同步化方法诱导获得龙眼体胚不同阶段材料,并以龙眼体细胞胚胎发生不同阶段混合材料作为试验材料,采用RT-PCR结合RACE技术分离并克隆龙眼中编码同源异型结构域蛋白的转录因子WUSCHEL(简称DlWUS)的cDNA全长及DNA序列,并进行序列分析与表达分析。结果表明:DlWUS的cDNA全长1 110bp,开放阅读框(ORF)858bp,共编码285个氨基酸(GenBank登录号为KM017506),DlWUS的DNA包含2个内含子。序列分析表明,DlWUS是一个不稳定的亲水蛋白,不含信号肽,亚细胞定位于细胞核,具跨膜结构和Homeodomain超级家族的保守结构域以及WUS转录因子家族特有的WUS box和EAR-like结构域,推测该目的基因确实为WUS转录因子。系统进化分析显示,龙眼DlWUS与脐橙WUS归为一个分支,亲缘关系较近。实时荧光定量PCR分析结果表明,在龙眼体细胞胚胎发生整个过程中,DlWUS均有表达,但仅在球形胚时期表达量较高,说明DlWUS可能主要在球形胚阶段发挥作用,并且在一定浓度范围内,外源施加IAA和GA3能够促进DlWUS基因的表达,而外源施加SA则抑制DlWUS基因的表达。