Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning,expression and substrate specificity analysis

Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3′- and 5′-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (l-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses.     

Molecular characterization and expression analysis of a complement component 3 in the sea cucumber (Apostichopus japonicus)

The complement system has been discovered in invertebrates and vertebrates, and plays a crucial role in the innate defense against common pathogens. As a central component in the complement system, complement component 3 (C3) is an intermediary between innate and adaptive immune system. In this study, a new isoform of C3 in the sea cucumber Apostichopus japonicus, termed AjC3-2 was identified. Its open reading frame (ORF) is 5085?bp and encodes for 1695 amino acids with a putative signal peptide of 20 amino acid residues. The mature protein molecular weight of AjC3-2 was 187.72?kDa. It has a conserved thioester site and a linker R(689)RRR(692) where AjC3-2 is splitted into β and α chain during posttranslational modification. The expression patterns of two distinct sea cucumber C3 genes, AjC3-2 and AjC3, were similar. During the different development stages from unfertilized egg to juvenile of the sea cucumber, the highest expression levels of AjC3-2 and AjC3 genes were both found in late auricularia. In the adult, the highest expression of these two genes was observed in the coelomocytes and followed by the body wall. AjC3-2 and AjC3 genes expression increased significantly at 6?h after the LPS challenge. These results indicated that these two C3 genes play a pivotal role in immune responses to the bacterial infection in sea cucumber.     

Identification and functional analysis of foxl2 and nodal in sea cucumber,Apostichopus japonicus

Sea cucumber (Apostichopus japonicus) is an important mariculture species in China. To date, the mechanisms of sex determination and differentiation in sea cucumber remain unclear. Identifying sex-specific molecular markers is an effective method for revealing the genetic basis of sex determination and sex differentiation. In this study, foxl2 and nodal homologous genes were identified in A. japonicus. Foxl2 exhibited dynamic and sexually dimorphic expression patterns in the gonads, with prominent expression in the ovaries and minimal expression in the testis according to real-time quantitative PCR (RT-qPCR) study. As nodal was specifically expressed in the ovary, it could serve as an ovary-specific marker in sea cucumber. Additionally, knockdown of foxl2 or nodal using RNA interference (RNAi) led to the down-regulation of piwi, germ cell-less, and dmrt1, suggesting that foxl2 and nodal may play important roles in gonad maintenance of sea cucumber. Overall, this study adds to our understanding of the roles of foxl2 and nodal in the gonadal development of A. japonicus, which provides further insight into the mechanisms of sea cucumber sex determination and differentiation.     

胶孢炭疽菌CgRGS2基因的克隆及生物学功能

【目的】G蛋白信号调控因子(Regulators of G-protein signaling,RGS)是G蛋白的一类负调控因子,在植物病原菌生长发育及致病过程中发挥着重要的作用,然而目前还未有关于胶孢炭疽菌RGS蛋白生物学功能的研究。本试验的目的是克隆胶孢炭疽菌的一个RGS基因CgRGS2,并分析其生物学功能。【方法】利用PCR技术扩增CgRGS2的基因并进行生物信息学分析,利用同源重组的方法获得CgRGS2基因的敲除突变体,并在突变体的基础上获得互补株,通过表型分析确定该基因的生物学功能。【结果】通过PCR扩增获得了CgRGS2的基因,其编码一个574个氨基酸的蛋白,在N末端含有一个RGS功能域。该基因敲除突变体同野生型相比,表现为营养生长缓慢,气生菌丝浓密,分生孢子产量降低且孢子呈多端萌发,对氧化压力及SDS敏感,致病性减弱等。【结论】CgRGS2蛋白参与调控胶孢炭疽菌的营养生长,分生孢子产量及萌发,氧化应激反应及细胞壁完整性,对其致病性也具有一定的影响。     

Phenotypic plasticity of gut structure and function during periods of inactivity in Apostichopus japonicus

Apostichopus japonicus is a common sea cucumber that undergoes seasonal inactivity phases and ceases feeding during the summer months. We used this sea cucumber species as a model in which to examine phenotypic plasticity of the digestive tract in response to food deprivation. We measured the body mass, gross gut morphology and digestive enzyme activities of A. japonicus before, during, and after the period of inactivity to examine the effects of food deprivation on the gut structure and function of this animal. Individuals were sampled semi-monthly from June to November (10 sampling intervals over 178 days) across temperature changes of more than 18 degrees C. On 5 September, which represented the peak of inactivity and lack of feeding, A. japonicus decreased its body mass, gut mass and gut length by 50%, 85%, and 70%, respectively, in comparison to values for these parameters preceding the inactive period. The activities of amylase, cellulase and lipase decreased by 77%, 98%, and 35% respectively, in comparison to mean values for these enzymes in June, whereas pepsin activity increased two-fold during the inactive phase. Alginase and trypsin activities were variable and did not change significantly across the 178-day experiment. With the exception of amylase and cellulase, all body size indices and digestive enzyme activities recovered and even surpassed the mean values preceding the inactive phase during the latter part of the experiment (October-November). Principal Component Analysis (PCA) utilizing the digestive enzyme activity and body size index data divided the physiological state of this cucumber into four phases: an active stage, prophase of inactivity, peak inactivity, and a reversion phase. These phases are all consistent with previously suggested life stages for this species, but our data provide more defined characteristics of each phase. A. japonicus clearly exhibits phenotypic plasticity (or life-cycle staging) of the digestive tract during its annual inactive period.     

Comparative analysis of genes expressed in regenerating intestine and non-eviscerated intestine of Apostichopus japonicus Selenka (Aspidochirotida: Stichopodidae) and cloning of ependymin gene

The regeneration of the intestine of sea cucumber (Apostichopus japonicus) was studied by describing historically the changes that occurred during intestine regeneration on the fifth day after chemically-induced evisceration. An expressed sequence tag (EST) analysis was undertaken to identify major genes, which might be involved in intestine regeneration of A. japonicus. Two cDNA libraries were constructed with directional cloning method, one for regenerating intestine collected on the third, fourth and fifth day after evisceration (post-evisceration, PE), and the other for the non-eviscerated (NE). A total of 730 ESTs were generated by sequencing cDNA clones from the two libraries (372 from PE and 358 from NE). The results showed that the number of genes that were involved in primary metabolism of PE library was less than that of NE library, while the number of genes involved in cell defense/immunity, cell division, cell signal transduction/communication of PE library was more than that of NE library. The results also revealed that the expression of the genes which might be involved in regeneration was enhanced to some extent after evisceration. Only about 11.54% of the sequenced clones were shared by two libraries, which provided some clues for the existence of differential gene expression between PE and NE intestines. A gene named epenAj was also characterized in this study.     

ARDRA分型测定刺参养殖环境中蛭弧菌多样性

【目的】研究刺参养殖环境中蛭弧菌类微生物的多样性及其组成特征。【方法】对刺参养殖用水进行过滤,获得微生物并提取其总DNA,分别采用两对特异性引物Per和Bac扩增蛭弧菌类微生物相应的16S r RNA基因目的片段。通过核糖体DNA扩增片段限制性内切酶分析(ARDRA)方法,使用HaeⅢ和MspⅠ,双酶切各60个目的基因的单克隆,选不同条带克隆进行测序并对测序结果的多重序列分析比对。【结果】确定两对引物分别存在8和9种碱基差异序列,均属于噬菌弧菌属,分属于3个类群中。类群Ⅰ、Ⅱ为优势类群,且均为已知类群,类群Ⅲ为潜在的新类群;Per1(KP214541)和Bac44(KP214551)代表菌株分别为优势种。【结论】刺参养殖环境中蛭弧菌具有较高的多样性,包括已知类型和未知类型的蛭弧菌;可进一步进行蛭弧菌的分离、培养,用于刺参养殖中病害防治研究。     

刺参甘露糖结合凝集素的生物信息学分析

甘露糖结合凝集素(mannan-binding lectin,MBL)属于C型动物凝集素超家族,特异地结合甘露糖。MBL是宿主起始免疫系统一个重要成分,因此近年来逐渐成为研究的热点。采用生物信息学技术对刺参(Apostichopus japonicus)MBL(AJ-MBL)的两个基因编码的蛋白质结构和功能进行了分析,包括信号肽、分子量、等电点、跨膜结构域、糖基化和磷酸化位点、二级结构和三级结构等,旨在了解其结构特征,为动物免疫反应方面研究提供理论基础。     

Gene cloning and function analysis of replication factor C from Thermococcus kodakaraensis KOD1

Replication factor C (RFC) catalyzes the assembly of circular proliferating cell nuclear antigen (PCNA) clamps around primed DNA, enabling processive synthesis by DNA polymerase. The RFC-like genes, arranged in tandem in the Thermococcus kodakaraensis KOD1 genome, were cloned individually and co-expressed in Escherichia coli cells. T. kodakaraensis KOD1 RFC homologue (Tk-RFC) consists of the small subunit (Tk-RFCS: MW=37.2 kDa) and the large subunit (Tk-RFCL: MW=57.2 kDa). The DNA elongation rate of the family B DNA polymerase from T. kodakaraensis KOD1 (KOD DNA polymerase), which has the highest elongation rate in all thermostable DNA polymerases, was increased about 1.7 times, when T. kodakaraensis KOD1 PCNA (Tk-PCNA) and the Tk-RFC at the equal molar ratio of KOD DNA polymerase were reacted with primed DNA.     

Molecular cloning and expression analysis of alpha 2-macroglobulin in the kuruma shrimp, Marsupenaeus japonicus

The cDNA encoding the kuruma shrimp, Marsupenaeus japonicus alpha(2)-macroglobulin (alpha(2)M) was obtained by screening a haemocyte cDNA library and 5' RACE PCR amplification. The full length cDNA of 4748 bp contains an open reading frame of 4518 nucleotides that translates into a 1505-amino acid putative peptide, with a 5'untranslated region (UTR) of 59 bp and a 3'UTR of 171 bp. The open reading frame encodes an N-terminal signal sequence of 17 residues and a mature protein of 1488 residues. The entire amino acid sequence is similar to the alpha(2)M sequences of arthropods (30-31% identity), mammals (26-27% identity) and fish (25-28% identity). The M. japonicus alpha(2)M sequence contains putative functional domains including a bait region, an internal thiol ester site, and a receptor-binding domain, which are present in mammalian alpha(2)Ms. In a healthy shrimp, the mRNA of alpha(2)M was mainly expressed in haemocytes. In addition, the expression level of alpha(2)M mRNA was dramatically increased by through time upon oral administration of peptidoglycan (PG), which is an immune stimulant. The highest expression of alpha(2)M mRNA was observed 7 days after feeding with PG. These results suggest that the shrimp alpha(2)M is an important molecule in immune system.     

仿刺参养殖池塘浮游病毒丰度与环境因子的相关性分析

为了揭示仿刺参养殖池塘生态系统中浮游病毒与环境因子的关系,于2008年3—11月对大连市谢屯地区的仿刺参养殖池塘中的浮游病毒丰度进行了定期检测,同时对水温、pH、溶解氧、盐度、叶绿素a含量、化学需氧量、无机氮、活性磷酸盐、异养细菌等因子进行了监测,对浮游病毒丰度与这些环境因子之间的相关性进行了分析。结果表明:仿刺参养殖池塘中浮游病毒的平均丰度为8.32×1010VLPs.L-1(最高值为4月的18.2×1010VLPs.L-1,最低值为11月的1.31×1010VLPs.L-1),外海水中浮游病毒平均丰度为6.45×1010VLPs.L-1(最高值为4月的12.6×1010VLPs.L-1,最低值为6月的2.02×1010VLPs.L-1),仿刺参养殖池塘中营养盐、水温、pH及盐度对浮游病毒丰度的影响较大,而外海水中叶绿素a和异养细菌对浮游病毒丰度的影响较大。     

日本对虾(Marsupenaeys japonicus)HsP70基因cDNA的克隆与序列分析

根据中国明对虾(Fenneropenaeuschinensis)Hsp70的cDNA序列及日本对虾(Marsupenaeusjaponicus)Hsp70cDNA序列片段设计引物,用RT-PCR方法,从经热休克处理的日本对虾鳃总RNA中克隆到长1992bp的Hsp70cDNA序列,包括长1959bp的完整编码序列(CDS)。根据编码序列推导出相应的652个氨基酸,与其他真核生物Hsp70家族成员进行同源性比较,发现与凡纳滨对虾(Litopenaeusvannamei)、斑节对虾(PinaeusmonodonFabricius)、中国明对虾(Penaeuschinesis)的相似度分别为99.5%、99.4%、99.4%,与日本沼虾(Macrobrachiumnipponense)和罗氏沼虾(M.rosenbergii)的相似度分别为93.7%和92.9%,与虹鳟(Oncorhynchusmykiss)、原鸡(Callusgallus)、家鼠(Musmusculus)和人(Homosapiens)的相似度为89.2%、85.4%、88.3%和88.3%,表现出较高的保守性。分析表明所得到的日本对虾Hsp70是一种Dnak亚家族类型的细胞质Hsp70,拥有完整的N端ATP酶结构域(约45kDa)、底物肽结合结构域(18kDa)及C端结构域(10kDa)。以上结果说明我们所得到的序列是一条包含完整CDS的Hsp70基因序列。     

仿刺参基因组DNA提取方法改进

开发了利用仿刺参管足活体取样提取基因组DNA的方法。按照传统方法提取仿刺参基因组DNA需要杀死实验对象并剖取体腔内纵肌(通常100mg)作为实验材料,并且实验过程中常常因为仿刺参的生物学性状而影响最终实验效果。以仿刺参在生活状态下拔取少量管足(10～20mg即可)作为取样源,在保持仿刺参的生活力不受影响的同时提高了取样效率。对仿刺参基因组DNA的提取方法进行了改进优化,并在常规海洋动物基因组DNA提取方法的基础上增加了部分操作步骤。与常规的基因组DNA提取方法相比,改进后的方法获取的基因组DNA完全符合后续实验要求。而且可随时根据即时效果(中间检测)和后续实验要求调整实验步骤,更增加了实验的可操作性。     

Identification and expression analysis of two Toll-like receptor genes from sea cucumber (Apostichopus japonicus)

Toll-like receptors (TLRs) are a family of type I integral membrane glycoproteins which play pivotal roles in innate immunity. In this study, two TLRs named AjTLR3 and AjToll were cloned from sea cucumber (Apostichopus japonicus). The full-length cDNA sequences of AjTLR3 and AjToll are 3484 bp and 4211 bp, with an open reading frame (ORF) of 2679 bp and 2853 bp, encoding 892 and 950 amino acids, respectively. Both AjTLR3 and AjToll are composed of a leucine-rich repeat (LRR) domain, a transmembrane (TM) domain and an intracellular Toll/interleukin-1 receptor (TIR) domain. Evolution analysis revealed that AjTLR3 and AjToll were clustered with the vertebrate-like TLRs (V-TLRs) and the protostome-like TLRs (P-TLRs), respectively. These two genes were widely expressed in all five tested tissues (body wall, coelomocytes, tube feet, intestine and respiratory tree), but showed different expression patterns. The significantly up-regulated expressions of AjTLR3 and AjToll after peptidoglycan (PGN), lipopolysaccharides (LPS), Zymosan A and polyinosinic–polycytidylic acid (PolyI:C) challenges suggested that they were functionally involved in the immune responses to the Cram-positive bacteria, Gram-negative bacteria, fungi and double-stranded RNA (dsRNA) viruses, respectively.     

Antioxidant and antihyperlipidemic activities of polysaccharides from sea cucumber Apostichopus japonicus

Polysaccharides (AJP) were prepared from Apostichopus japonicus by protease hydrolysis method. Antioxidant activity in vitro and antihyperlipidemic activity in vivo was investigated. Chemical composition analysis indicated that AJP was mainly composed of glucosamine, galactosamine, glucuronic acid, mannose, glucose, galactose and fucose, with an average molecular weight of about 36.2kDa. The antioxidant capacities of AJP were, respectively, evaluated by the assays of scavenging DPPH, hydroxyl and superoxide radicals, and reducing power in vitro. It showed potent free radical scavenging activities and reducing power. Serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL-C) decreased significantly and high-density lipoprotein cholesterol (HDL-C) increased significantly after treatment of hyperlipidemic Wistar rats with AJP. These results suggest that AJP may prove to be a potential candidate of the natural antioxidants as a therapeutic agent for hyperlipidemia.