Sequence and structural analysis of 4SNc-Tudor domain protein from Takifugu Rubripes

The fugu SN4TDR protein belongs to an evolutionarily conserved family, consisting of four repeat staphylococcal nuclease-like domains (SN1-SN4) at the N-terminus followed by Tudor and SN-like domains (TSN). Sequence analysis showed that the C-terminal TSN domain is composed of a complete SN-like domain interdigitated with a Tudor domain. In despite of low level of sequence identities, five SN-like domains have a few conserved amino acids that may play essential roles in the function of the protein. Computer modeling and secondary structural prediction of the SN-like domains revealed the presence of similar structural features of β1-β2-β3-α1-β4-β5-α2-α3, which provides a structural basis for oligonucleotides binding. The loop region L_3α for binding sites between β3 and α1 of SN-like domains are different from human p100, implying the divergence in the structures of binding sites. These results indicate that fugu SN4TDR may bind methylated ligands and/or oligonucleotides through its distant domains.     

Molecular characterization and transcriptional analysis of non-mammalian type Toll like receptor (TLR21) from rock bream (Oplegnathus fasciatus)

Toll-like receptors (TLRs) are a large family of pattern recognition receptors, which are involved in triggering host immune responses against various pathogens by detecting their evolutionarily conserved pathogen associated molecular patterns (PAMPs). TLR21 is a non-mammalian type TLR, which recognizes unmethylated CpG DNA, and is considered as a functional homolog of mammalian TLR9. In this study, we attempted to identify and characterize a novel TLR21 counterpart from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at molecular level. The complete coding sequence of RbTLR21 was 2919 bp in length, which encodes a polypeptide of 973 amino acids with a predicted molecular mass of 112 kDa and a theoretical isoelectric point of 8.6. The structure of the deduced RbTLR21 protein is similar to that of the members of typical TLR family, and includes the ectodomain, which consists of 16 leucine rich repeats (LRRs), a transmembrane domain, and a cytoplasmic Toll/interleukin-1 receptor (TIR) domain. According to the pairwise sequence analysis data, RbTLR21 was homologous to that of the orange-spotted grouper (Epinephelus coioides) with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 is closely related to E. coioides TLR21. The RbTLR21 was ubiquitously expressed in all the tissues tested, but the highest expression was found in spleen. Additionally, upon stimulation with Streptococcus iniae, rock bream iridovirus (RBIV), and Edwardsiella tarda, RbTLR21 mRNA was significantly up-regulated in spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed an ortholog of the TLR21 family and may be important in mounting host immune responses against pathogenic infections.     

Involvement of host recognition by oncomiracidia and post-larval survivability in the host specificity of Heterobothrium okamotoi (Monogenea: Diclidophoridae)

Heterobothrium okamotoi, a monogenean gill parasite, shows high host specificity for tiger puffer, Takifugu rubripes. In the present study, in vivo and in vitro experimental infections were conducted using various fish species, including T. rubripes, to understand the mechanisms of specificity. In in vivo experiments, T. rubripes, grass puffer, Takifugu niphobles, olive flounder, Paralichthys olivaceus, and red sea bream, Pagrus major, were exposed to oncomiracidia of H. okamotoi labelled with a fluorescent dye, 5- (and -6) carboxyfluorescein diacetate succinimidyl ester, and the numbers of parasites on the gills and skin were recorded at intervals. Oncomiracidia were attached to gills and skin of all the experimental fish species immediately after exposure, and the infection intensity on T. rubripes was higher than that on T. niphobles and much higher than those on the other two species. After 2 days, the attached parasites remained on the gills of T. rubripes, but disappeared from the other hosts. During in vitro experiments, gill filaments excised from seven different fish species (four fish species used in the in vivo experiments and panther puffer, Takifugu pardalis, southern flounder, Paralichthys lethostigma and spotted halibut, Verasper variegates) were exposed to oncomiracidia and the attachment to each fish species and subsequent larval behaviour was observed. The percentage of post-larvae that attached to T. rubripes was slightly higher than those which attached to congeneric fish species and much higher than those of non-tetraodontid fish species. In vivo and in vitro experiments demonstrated that oncomiracidia of H. okamotoi have an affinity for their natural host, T. rubripes, and congeneric fish species. The disappearance of attached post-larvae from 'alien' hosts within 2 days during in vivo experiments suggested that host recognition by oncomiracidia and subsequent post-larval survivability are involved in the host specificity of H. okamotoi.     

鄱阳湖野生河豚鱼体内河豚毒素产生菌的分离鉴定(英文)

从中国鄱阳湖捕获的暗纹东方鲀卵巢中分离到4株菌,通过小鼠实验、薄层色谱、质谱分析及荧光分光光度法确认TL-1菌株发酵液中含河豚毒素。24℃培养5 d,通过活性炭柱、凝胶柱从TL-1发酵液中分离河豚毒素粗毒液。经形态、生理生化及16S rRNA分析鉴定,表明该菌属于花域芽孢杆菌属。本研究首次报道从鄱阳湖河豚鱼中分离到产毒菌。     

暗纹东方鲀耐寒相关基因CIRBP、HMGB1和AFP-Ⅳ的分子特征和对低温胁迫的响应

为了探索暗纹东方鲀(Takifugu obscurus)对低温环境的响应机制,克隆了暗纹东方鲀耐寒相关基因CIRBP、HMGB1和AFP-Ⅳ的cDNA序列,并进行了基因的分子特征和功能分析。组织分布检测显示CIRBP和HMGB1在下丘脑、肝脏和肌肉中具有高表达,而AFP-Ⅳ则主要在肝脏中表达。在受到低温胁迫后, 3种基因在肝脏和下丘脑中的表达呈现不同的变化趋势,其中CIRBP基因在肝脏中于48h表达量显著增加,在下丘脑中于12h和48h有上调表达; HMGB1基因在肝脏中呈现逐渐上升的趋势,于48h达到最大值,而在下丘脑中呈现先上升后下降的趋势,处理后2h达到最大, 2—8h下降,于8h下降至最低,随后恢复至初始水平;肝脏中的AFP-Ⅳ在0—24h无显著变化,在48h上升至最大值。进一步通过大肠杆菌原核表达系统研究了AFP-Ⅳ的抗冻功能,发现AFP-Ⅳ融合蛋白在–80℃下具有抗冻活性,并且抗冻活性随着浓度的增加而提高。研究结果表明3种基因都参与了暗纹东方鲀对低温胁迫的应答过程,为深入探索暗纹东方鲀的耐低温机制奠定了基础。     

菊黄东方鲀、暗纹东方鲀及其杂交F1代肌肉营养成分的比较分析

为了了解菊黄东方鲀(Takifugu flavidus)、暗纹东方鲀(T.obscurus)及其杂交F1代的肌肉营养特征,利用生物化学方法,从每类实验样本中取9尾对其肌肉中的粗蛋白、粗脂肪、水分、粗灰分和氨基酸成分进行了测定和分析。结果显示:(1)杂交F1代在生长方面具有明显的杂交优势,与亲本之间存在着显著差异(P0.05),杂交F1代的体重为其亲本的1.48~1.77倍;(2)杂交F1代肌肉水分含量与其母本含量相近,但粗脂肪含量均较亲本少(P0.05),粗蛋白含量则与亲本差异不显著(P0.05);(3)除色氨酸和胱氨酸外,16种氨基酸均在肌肉样本中被检测到,除甲硫氨酸外,其余15种氨基酸间含量均存在着显著性差异(P0.05)。菊黄东方鲀(♀)×暗纹东方鲀(♂)杂交F1代的总氨基酸含量最高,而暗纹东方鲀(♀)×菊黄东方鲀(♂)F1代总氨基酸含量则介于两亲本之间。对其必需氨基酸总量进行分析发现,菊黄东方鲀与其正反杂交F1代之间均存在显著性差异(P0.05),而暗纹东方鲀与其正反杂交F1代之间差异不显著(P0.05);(4)肌肉营养品质评价结果表明,菊黄东方鲀(♀)×暗纹东方鲀(♂)杂交F1代的鲜味氨基酸含量为26.68%,明显高于双亲样本(菊黄东方鲀22.28%、暗纹东方鲀25.20%),而暗纹东方鲀(♀)×菊黄东方鲀(♂)F1代的鲜味氨基酸总量(23.30%)较其父本偏高,但低于其母本。研究结果表明,杂交东方鲀的肌肉营养综合了双亲的优良特性,特别是是菊黄东方鲀(♀)×暗纹东方鲀(♂)杂交F1代,拥有最高的鲜味氨基酸含量,具有推广价值。     

暗纹东方鲀侧线系统早期形态和生长发育

通过光镜和扫描电镜对暗纹东方鲀(Takifugu obscurus)的侧线系统进行形态学及组织学的研究。研究结果首次揭示了暗纹东方鲀侧线系统除了主侧线外还包括辅助侧线和辅助神经丘。主侧线分布主要包括眶上线、眶下线、耳后侧线、下颌线、前鳃盖线、上颞线、背侧线、腹侧线。辅助侧线和辅助神经丘分布主要包括口部辅助侧线、眶下辅助侧线、下颌前辅助侧线、下颌后辅助侧线、眶上后辅助侧线、上颞腹辅助神经丘、上颞背辅助神经丘、前鳃盖后辅助神经丘、背部辅助神经丘、尾部辅助神经丘。暗纹东方鲀侧线器官为接受机械刺激的神经丘,数目上千,神经丘分布在体表的凹槽里,且位于高低不同突起顶端。神经丘由套细胞、支持细胞和感觉毛细胞组成。感觉毛细胞呈圆形排列,并且每个细胞的游离面均有一根动纤毛和几十根静纤毛。据本研究对暗纹东方鲀侧线分布特征和神经丘的生长特征等的观察结果,认为尽管暗纹东方鲀侧线系统没有如其他真骨鱼类的管道系统,但是依然具有两套不同生理机能的机械感受系统,符合"七管模式"的主侧线神经丘与管道神经丘同源,而辅助侧线和辅助神经丘才是真正的表面神经丘。     

袋鼠花水培扦插技术研究

扦插是袋鼠花繁殖的主要方法。本实验采用不同剂量营养液与吲哚丁酸(IBA)不同浓度相结合的方法,以清水为对照,通过测定袋鼠花的生根情况,探讨袋鼠花水培扦插的适宜处理组合。结果表明:营养液方面,清水处理对根条数、根长、根鲜重和生根率的影响明显优于其它处理,有利于袋鼠花的水培生根;IBA浓度为20mg/L时的生根率明显优于其它处理和对照,而当其浓度为40mg/L时对于根条数、根长和根鲜重则明显优于对照和其它处理,最有利于袋鼠花的水培生根。实验结果说明较低浓度的营养液(如清水)与中等浓度(如40mg/L)的IBA激素最有利于袋鼠花的水培生根。因此,本研究为缩短水培袋鼠花的生产周期及突出其观根特色,提高其观赏价值和商品价值提供理论依据。