口虾蛄卵黄蛋白原(Vg)基因的初步研究

为研究口虾蛄Oratosquilla oratoria卵黄蛋白原(vitellogenin,Vg)基因,利用c DNA末端快速扩增(RACE)技术克隆口虾蛄Vg基因c DNA全长序列(Gen Bank登陆号:KR422400),并应用相对实时荧光定量PCR技术检测雌雄口虾蛄不同组织、不同发育时期卵巢Vg mRNA的表达量。结果表明:口虾蛄Vg基因全长7727 bp,其中5'端非编码区为36 bp,开放阅读框(ORF)为7521 bp,3'端非编码区为170 bp,共编码2506个氨基酸;预测的氨基酸序列中存在1个长度为20个氨基酸的信号肽,1个类似于枯草蛋白酶的内切蛋白酶识别位点(RSKR),3个潜在的N-连接糖基化位点,1个在无脊椎和脊椎动物卵黄蛋白原中都比较保守的KALGNVG基序;对其结构域分析表明,口虾蛄Vg蛋白含有4种保守结构域,分别为卵黄蛋白原N端结构域(Vitellogenin_N)、血管性血友病因子(v WFD)和两种未知功能结构域(domain of unknown function)DUF1943与DUF1081;经NCBI BLASTP同源性比较,口虾蛄Vg氨基酸序列与其他甲壳类的相似性为46%~52%;系统发育分析结果显示,口虾蛄Vg单独聚为一支;实时定量PCR结果显示,口虾蛄Vg基因在性腺和肝胰腺中均有表达,在肌肉中均不表达,卵巢中Vg mRNA的表达量显著高于其他组织(P0.05),且口虾蛄Vg mRNA的相对表达量随着卵巢的发育不断增加(P0.05),在成熟期时达到最高值,随后显著下降,Vg mRNA表达量在卵巢中的变化规律可以作为了解口虾蛄性腺发育的有效指标。本研究结果为口虾蛄Vg蛋白的功能研究奠定了基础。     

Improved vitellogenesis,gonad development and egg diameter in catfish (Pangasianodon hypophthalmus) supplemented with turmeric (Curcuma longa) powder

An experiment was conducted to study the effect of supplementing catfish (Pangasianodon hypophthalmus) with turmeric (Curcuma longa) on the vitellogenic capacity of the liver as a new method to improve reproductive performance in oviparous animals. The experimental catfish were assigned to a completely randomized design consisting of four doses of supplemental turmeric (0, 1.2, 2.4 and 4.8 g/kg feed) with 10 catfish with body weights ranging from 2 to 4 kg in each group. Blood samples were collected on days 0, 14, 28, 42 and 56 of turmeric supplementation to measure the concentrations of plasma oestradiol‐17ß and vitellogenin. The weights of the body, gonads and liver were measured on days 0, 28 and 56. The fecundities of the catfish were measured on days 28 and 56 of turmeric supplementation, and egg diameters were measured on days 28 and 42. The results showed that catfish supplemented with turmeric at a dose of 2.4 or 4.8 g/kg feed had higher growth rates, higher plasma oestradiol‐17β and vitellogenin concentrations, and higher gonad somatic index values and egg diameters. This is the first report demonstrating that the hepatoprotective activity of the curcumin in turmeric could be used to improve both vitellogenin synthesis, which improves nutrient deposition in the ovulating eggs, and the reproductive performance of teleost fish and oviparous animals.     

Effects of dietary soy isoflavones on estrogenic activity,cortisol level,health and growth in rainbow trout,Oncorhynchus mykiss

Soy isoflavones (the phytoestrogens genistein, daidzein and glycitein) may act as estrogen receptor agonists or antagonists. The aim of this study was to examine the effect of dietary isoflavones on growth, reproduction and health in rainbow trout (Oncorhynchus mykiss). Rainbow trout fed three experimental diets containing different concentrations of isoflavones (0, 500 and 1,500 ppm). Growth, estrogenic activity, plasma estradiol levels, gonadal development, state of stress and histological changes in selected tissues were evaluated at the end of 70 days. Neither growth performance nor the relative mRNA levels of Insulin Growth Factor I (igf‐I) in the liver were influenced by different levels of dietary isoflavones. Plasma and liver vitellogenin (VTG) protein levels and plasma 17‐β‐estradiol (E₂) were unaffected by treatments, although the correlation between plasma levels of E₂ and VTG densitometry values was significant (p < .05). The fish gonadosomatic index (GSI) did not significantly differ among the three experimental groups but correlated with plasma VTG densitometry values (p < .05). Plasma, muscle and fin cortisol concentrations fell within the normal welfare range and were not correlated with isoflavone levels. Histologically, the distal intestine showed a normal morphology with well‐differentiated enterocytes and in the liver hepatocytes were also normal. A supranuclear accumulation of lipid droplets in enterocytes and some lipid droplets in hepatocytes were observed in all tested groups, suggesting an impact of basal dietary lipid on transport/metabolism of fat in the fish. Overall, the present results suggest that the doses of isoflavones tested do not compromise rainbow trout reproduction, growth and health.     

Effects of dietary soybean lecithin on gonadal development and vitellogenin mRNA expression in the female redclaw crayfish Cherax quadricarinatus (von Martens) at first maturation

The effect of increasing levels of dietary de‐oiled soybean lecithin (SL) on the ovarian development of female redclaw crayfish, Cherax quadricarinatus, was investigated. Five practical formulated diets were supplemented with 0% (Diet 1), 1% (Diet 2), 2% (Diet 3), 4% (Diet 4) and 6% (Diet 5) SL. Crayfish (initial weight: 25.64 ± 1.53 g) were fed each diet in four replicates for 8 weeks. Crayfish that were fed diets containing ≥2% SL had a significantly higher gonadosomatic index than those fed with Diets 1 and 2 (P < 0.05), while hepatosomatic index showed a decreasing trend (P > 0.05). The fatty acid composition of the hepatopancreas is largely due to the dietary composition. Higher dietary SL yielded a higher percentage of ovarian polyunsaturated and highly unsaturated fatty acids (P < 0.05). Furthermore, the higher content of linoleic acid and alpha linolenic acid in diets may be more important for ovarian development in C. quadricarinatus, as these originate from the hepatopancreas. The hepatopancreatic vitellogenin mRNA expression was the highest in crayfish that received Diet 3. Our results suggest that dietary SL has a positive effect on ovarian development in C. quadricarinatus broodstock, and at least 2% SL should be supplemented in broodstock diets to enhance ovarian maturation during aquaculture.     

Environmental control of fish reproduction: a different perspective

Nearly all studies on the environmental control of fish reproduction have, to date, focused on the roles played by various parameters of the natural environment, such as changes in water temperature and photoperiod. While these factors undoubtedly still play significant roles, other factors, which have arisen as a consequence of man's activities, also play major roles in at least some aquatic environments. One such factor is pollution; whereas attention is usually focused primarily on severe pollution and the fish mortality that can result, recently some emphasis has been given to the more subtle, but potentially serious, consequences of less severe pollution. Many freshwater habitats, particularly rivers in urban areas, now receive substantial volumes of effluent from various sources (both industrial and domestic). This effluent can contain chemicals capable of mimicking endogenous hormones, and hence has the potential to disrupt endocrine-mediated processes such as reproduction. This paper is concerned with the identity of these endocrine-disrupting chemicals, their mechanisms of action, and the effects they have on reproduction, both in wild fish and in fish maintained in the laboratory.     

稀有鮈鲫卵黄蛋白原基因全序列的克隆与分析

稀有鮈鲫（Gobiocypris rarus）是我国特有的一种小型鲤科鱼类,目前已作为一种新型模式生物应用于实验研究。运用普通PCR方法克隆得到一段长6 299 bp的序列,序列分析发现编码区与稀有鮈鲫卵黄蛋白原cDNA序列相似性为100%。再参考6 299 bp序列5′端设计两对特异性引物,进行基因组步移,获得长度为1 285 bp的序列。经过序列拼接后得到7 573 bp的序列,分析发现其中含卵黄蛋白原基因全序列长度为 6 595 bp卵黄蛋白原基因上游调控序列长度为978 bp。进一步分析开放性阅读框得知：稀有鮈鲫卵黄蛋白原基因含有26个外显子和25个内含子,编码1 299个氨基酸序列,CDS长3 900 bp。经BLAST比对发现,稀有鮈鲫卵黄蛋白原上游调控序列中220～838 bp区间与朝鲜（Acheilognathus yamatsutae）卵黄蛋白原基因启动子序列2 857～3 469 bp 区间有80 %相似性。     

中华绒螯蟹卵黄蛋白原基因cDNA序列的克隆与结构分析

通过RT-PCR,RACE等技术克隆得到中华绒螯蟹卵黄蛋白原的cDNA序列全长,其长度为7 921 bp,软件分析后知其含有一个7 689 bp的开放阅读框,其5′和3′的非翻译区分别为28 bp和204 bp; 预测该序列编码一个含2 562个氨基酸残基的蛋白质,和其它已知卵黄蛋白原序列的蟹类相比,理化性质、空间构型相似,且含有2个结构域：参与脂质转运的卵黄蛋白原N端结构域（Vitellogenin_N）,以及血管性血友病因子（von Willebrand factor）D型结构域（VWD）。将其开放阅读框翻译为氨基酸序列后进行数据库对比（BLASTP）,与其他已知的甲壳类卵黄蛋白原序列间存在33%～77％的相似性。