暗纹东方鲀线粒体DNA控制区结构和系统发育分析

以暗纹东方(Takifugu fasciatus)肝脏线粒体DNA为模板,参照GenBank中红鳍东方(Takifugu rubripes)线粒体DNA序列设计合成特异引物进行PCR扩增,获得了暗纹东方线粒体DNA控制区基因(818 bp)及5′端上游的tR-NAPro基因(71 bp)的全序列。控制区碱基组成为T32.2%,C 19.1%,A35.6%,G13.2%。对照其他已报道的鱼类控制区结构,对暗纹东方控制区的结构进行了分析,识别了其终止序列区、中央保守区和保守序列区,找到了终止相关的序列TAS以及保守序列(CSB1,CSB2,CSB3)。CSB1、CSB2序列相对保守,TAS与其回文基序可形成稳定的茎环结构,成为H-链复制延伸时的终止识别位点。同时运用DNA分析软件对暗纹东方与GenBank中其他10多种鱼类的mtDNA控制区序列进行比对,并选取东方属的7种鱼类mtDNA控制区序列构建分子系统树。结果显示控制区基因较适于科鱼类中同属不同种的系统发育分析。     

哈氏弧菌胞外产物对红鳍东方鲀的致病性

采用平板玻璃纸覆盖技术提取哈氏弧菌(Vibrio harveyi)H-06091菌株的胞外产物(ECP),通过肌肉和腹腔注射2种方式研究其对红鳍东方鲀(Fugu obscurus)的致病性。采用双层纸片法对其主要酶成份进行初步研究;测定其对绵羊红细胞和红鳍东方鲀红细胞的溶血活性;将哈氏弧菌在不同温度下(15℃、20℃、25℃、30℃、35℃、40℃)培养36h和25℃下培养不同时间(12h、18h、24h、36h、48h、60h、72h)后提取其胞外产物并对其蛋白质含量进行测定;经SDS-PAGE分离粗提胞外产物中蛋白成分,并用Dot-ELISA和Western-blot方法对其成分进行分析。实验结果显示,该胞外产物对红鳍东方鲀具有致病性;具有淀粉酶和酪蛋白酶活性,不具有卵磷脂酶、脂肪酶和明胶酶活性;对红鳍东方鲀红细胞有溶血活性,对绵羊红细胞无溶血活性;胞外产物蛋白质总含量在35℃培养36h后出现高值,与温度变化无明显相关性;25℃下培养24h、36h和48h的粗提胞外产物经SDS-PAGE分离,有3条蛋白质条带最明显,分子量分别约为31kD、38kD、60kD;其中分子量约为60kD蛋白条带,经Dot-ELISA和Western-blot方法证实其为半胱氨酸蛋白酶-1。根据以上结果认为哈氏弧菌H-06091菌株胞外产物含有多种毒力因子,与该菌的致病性密切相关。     

暗纹东方鲀Amhr2基因外显子9的SNP7271检测及其与性别相关分析

利用传统PCR和ABI 3730测序技术对暗纹东方鲀(Takifugu obscurus)Amhr2基因外显子9进行了SNP7271检测和测序分析,并对该SNP位点与暗纹东方鲀性别相关性进行了研究。结果显示,在SNP7271位点发生了G/C碱基突变,产生了CG、CC两种基因型。SNP7271和暗纹东方鲀性别存在显著相关性(P0.01)。基因型为CG的个体生理性别为雄性,基因型CC的个体生理性别为雌性。     

菊黄东方鲀鱼精蛋白提取工艺优化研究

为研究河鲀鱼精蛋白的制备工艺,以菊黄东方鲀(Takifugu Flavidus)精巢为原料,以鱼精蛋白提取率为指标,通过单因素试验研究NaCl溶液的pH、硫酸浓度、硫酸用量、提取温度、提取时间以及乙醇用量等单因素对鱼精蛋白提取率的影响,在此基础上,采用响应面法优化鱼精蛋白的提取工艺条件。结果显示,采用响应面法优化获得河鲀鱼精蛋白的最佳提取工艺条件为:NaCl溶液pH 8.5,硫酸浓度0.6 mol/L,硫酸用量4.0倍沉淀体积,提取温度45℃,提取时间2.4 h,乙醇用量4.0倍沉淀体积,此时鱼精蛋白提取率达到19.07%。研究表明,响应面法对河鲀鱼精蛋白提取条件的优化方案合理可行,可为河鲀精巢的开发利用提供参考依据。     

Induced Ovulation in Obscure Puffer Takifugu obscurus by Injections of LHRH-a

Obscure puffer Takifugu obscurus (Abedate) is one of the several species of Takifugu genus that migrate to the Yangtze River, China during the spawning season of February to May. It is a popular fish among locals and fetches a premium price owing to its high-quality meat. It is also a potential species for intensive culture as its natural populations are currently declining. There have been no published studies on the effects of GnRH-a injection on the obscure puffer. In this paper, we report the results of several obscure puffer ovulation trials conducted in 1995 and 1996. We demonstrated that ovulation could successfully be induced in obscure puffer collected from open systems during spawning period using a dosage of LHRH-a as low as 40 µg kg^–1 in two injections. We found no significant differences among 80, 60, 40 µg LHRH-a kg^–1 treatments in ovulation success, egg production, fertilization rate and hatch rate. Accordingly, we recommend the use of 40 µg LHRH-a kg^–1 as the optimal dosage from an economical point of view.     

Histopathological studies on Vibrio harveyi– infected tiger puffer,Takifugu rubripes (Temminck et Schlegel), cultured in Japan

Vibrio harveyi infection occurred with a moderate mortality in tiger puffer, Takifugu rubripes (Temminck et Schlegel), in autumn 2007, at a mariculture farm in western Japan. The diseased fish showed nodular lesions in the branchial chamber and the inner surface of the operculum. Histopathologically, the lesions comprised granulation tissue containing many suppurative foci allowing propagation of the bacteria and granuloma encapsulating abscesses with a decrease in bacteria. The bacteria were disseminated in visceral organs including the spleen, kidney, liver, and myocardium, resulting in the formation of granulomatous lesions. Two groups of tiger puffer juveniles were artificially infected by an intramuscular injection with an isolate (1.0_10⁸ CFU/fish). During the experimental period, 20% mortality occurred within 4–6 days post‐infection (d.p.i). The fish sampled on 4 d.p.i showed abscesses in the lateral musculature at the injection site. The fish sampled 5 d.p.i. displayed the production of granulation tissue containing many suppurative foci, which replaced the necrotic dermis and lateral musculature. Surviving fish (15 d.p.i.) had granulomatous lesions in the lateral musculature at the injection site. Pyogranulomatosis is pathognomonic in V. harveyi infection of tiger puffer.     

集约化养殖条件下暗纹东方魨生长特征的研究

报道了在集约化养殖条件下４～２０月龄暗纹东方的生长特征。其中体长、体重的相对生长率和生长指标皆随月龄增加而逐渐下降。体长与体重呈指数曲线相关，方程为，体长、体重的生长方程分别为Ｌ＝１１９．５６Ｌｎ（ｔ）－９０．４６９，Ｗ＝０．４１２７ｔ。试验表明，暗纹东方纯在集约化养殖条件下具有良好的生长潜力，可进行规模化商品鱼养殖。     

壳聚糖和益生菌对暗纹东方鲀抗病力和免疫功能的影响

为了解壳聚糖和益生菌刺激和增强免疫应答的可能作用,本试验在基础饲料中添加0.2%、0.5%、1.0%的壳聚糖,0.1%、0.2%、0.4%的益生菌,甘露聚糖与益生菌混合物,壳聚糖与益生菌的混合物,在室内水泥池中喂养暗纹东方鲀(3.15±0.05)g 2个月,根据抗菌活力筛选出免疫增强饲料组(0.2%壳聚糖,0.1%、益生菌,甘露聚糖与益生菌混合物,壳聚糖与益生菌混合物)测定免疫功能。结果表明,甘露聚糖与益生菌混合物仅提高头肾T淋巴细胞转化,壳聚糖与益生菌混合物显著增强头肾T、B淋巴细胞转化和脾脏B淋巴细胞体外培养时分泌的IgM。筛选出的四种免疫增强饲料均能使头肾B淋巴细胞体外培养时分泌的IgM显著增加。无论是脾脏B淋巴细胞还是头肾B淋巴细胞,体外培养时所分泌的IFN-α都不同程度地受抑制。     

光照强度对暗纹东方鲀稚鱼摄食的影响

在0～1000lx光照范围内,对不同光照强度下暗纹东方鲀稚鱼摄食量进行了测定,稚鱼在500lx时摄食强度最大,其次为300、700lx,摄食率在500lx时最高,并在5min时达到最大值,40～60min时段摄食效率最低接近停食。     

A Novel Angiotensin-I-Converting Enzyme (ACE) Inhibitory Peptide from Takifugu flavidus

Alcalase, neutral protease, and pepsin were used to hydrolyze the skin of Takifugu flavidus. The T. flavidus hydrolysates (TFHs) with the maximum degree of hydrolysis (DH) and angiotensin-I-converting enzyme (ACE)-inhibitory activity were selected and then ultra-filtered to obtain fractions with components of different molecular weights (MWs) (<1, 1–3, 3–10, 10–50, and >50 kDa). The components with MWs < 1 kDa showed the strongest ACE-inhibitory activity with a half-maximal inhibitory concentration (IC₅₀) of 0.58 mg/mL. Purification and identification using semi-preparative liquid chromatography, Sephadex G-15 gel chromatography, RP-HPLC, and LC–MS/MS yielded one new potential ACE-inhibitory peptide, PPLLFAAL (non-competitive suppression mode; IC₅₀ of 28 μmmol·L⁻¹). Molecular docking and molecular dynamics simulations indicated that the peptides should bind well to ACE and interact with amino acid residues and the zinc ion at the ACE active site. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that PPLLFAAL could significantly decrease the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHRs after intravenous administration. These results suggested that PPLLFAAL may have potential applications in functional foods or pharmaceuticals as an antihypertensive agent.