黄鳝细胞质动力蛋白3基因的克隆及其在性逆转中的功能分析

为解析黄鳝(Monopterus albus)性逆转机制，实验以雌、雄、间性发育黄鳝为研究对象，分析不同组织、不同发育时期性腺、甲基睾丸酮处理性腺及Zebularine处理性腺原代细胞后dynlt3基因表达模式的变化及其在性腺中的表达定位。性腺转录组测序结果显示，基因全长1 082 bp,开放阅读框354 bp,编码117个氨基酸。生物信息学分析显示，dynlt3基因编码蛋白质的二级结构包含37.96%的α-螺旋，29.20%的β-折叠，32.85%的无规则卷曲。系统进化分析结果显示，黄鳝DYNLT3氨基酸序列与硬骨鱼纲中底鳉(Fundulus heteroclitus)同源性最高。实时荧光定量PCR结果表明，dynlt3基因在黄鳝肌肉和脑具有较高表达，心脏次之，在其它各组织表达量较低。在性腺的不同发育时期，在间性后期和雄性中表达量显著性高于雌性与间性早期。甲基睾酮处理后黄鳝卵巢组织结构发生明显退化，卵母细胞退化且数量减少，结缔组织间出现空泡结构；dynlt3基因在卵巢中表达量显著性下调。原位杂交分析dynlt3基因在性腺组织中的表达定位结果显示，在不同性腺发育时期均检测到dynlt3...     

泰国斗鱼Dmrt1基因的部分cDNA克隆及表达分析

Doublesex和Mab-3相关转录因子1(Dmrt1)隶属于DMRT家族,在脊椎动物性别决定、性别分化和性腺发育中具有重要作用。利用RACE克隆技术从泰国斗鱼脑中克隆Dmrt1基因的部分cDNA序列,分别采用半定量PCR和实时荧光定量PCR检测Dmrt1基因的组织分布和胚胎发育过程的表达变化。试验结果显示,克隆的泰国斗鱼Dmrt1基因的部分cDNA序列长为1037 bp,其中开放阅读框长为798 bp,编码265个氨基酸残基;进一步构建Dmrt1系统进化树,结果显示,泰国斗鱼与龟壳攀鲈亲缘关系最近;半定量PCR和实时荧光定量PCR结果显示,Dmrt1基因的表达具有明显的组织特异性及性别差异,在雄性个体中,Dmrt1基因在精巢和脾脏中特异表达,而在雌性个体中,Dmrt1基因在心脏中高表达,其次在脑、垂体、脾脏、肌肉、肾脏和肠道中也有少量表达,但未在性腺和肝脏中表达。在胚胎发育过程中,Dmrt1基因在受精卵的表达水平最高,而在原肠胚至心跳期表达量较低。Dmrt1可能在调控泰国斗鱼的雄性个体的生殖发育过程中发挥着重要作用。     

黄喉拟水龟性别调控相关lncRNA和mRNA的筛选及初步分析

性别异形在动物界广泛存在,长期以来一直是生物学中的热门话题。黄喉拟水龟性别分化属温度依赖型,其温度响应分子机制仍不清楚。为了进一步解析龟鳖动物性别分化的分子机制,本研究初步比较分析了黄喉拟水龟转录本中性别差异表达(different expressed, DE)的长链非编码RNA(long noncoding RNA, lncRNA)及其调控的靶基因。首先,运用Illumina深度测序平台,通过转录组测序(RNA-Seqs)对黄喉拟水龟的精巢和卵巢进行了比较转录组学分析并筛选鉴定出雌雄差异转录本,共筛选获得8 237个DE mRNA和9 573个DE lncRNA。通过GO功能注释及 KEGG通路富集分析发现,上述差异转录本主要参与龟鳖动物性别分化和性腺发育等相关信号通路。此外,通过顺式和反式作用分析,筛选获得了一系列与生殖发育相关的(性腺发育和性别分化)受DE lncRNAs调控的靶基因。本研究为进一步阐明黄喉拟水龟温度依赖型性别的分子机制提供了线索,特别是为进一步挖掘利用龟鳖动物性别决定因子,进行龟鳖动物性控育种技术研究奠定了基础。     

刺参响应灿烂弧菌侵染的基因组DNA甲基化水平和转录组差异及其关联分析

为探讨病原菌胁迫下刺参(Apostichopus japonicus)基因组DNA甲基化水平和转录组表达的差异,本研究采用人工攻毒侵染胁迫,获得刺参化皮体壁组织,并以未攻毒组的健康体壁组织为对照,对刺参2种体壁组织进行全基因组甲基化测序(WGBS)和转录组高通量测序,解析刺参体壁基因组DNA甲基化差异,筛选响应病原胁迫的差异甲基化区域和差异表达基因。同时,通过基因组甲基化和转录组联合分析,筛选负相关关联基因,为解析刺参响应灿烂弧菌(Vibrio splendidus)侵染的分子机理提供基础数据。结果显示,刺参对照组和侵染组基因组总甲基化水平分别为(3.59±0.04)%和(3.87±0.27)%,侵染组甲基化水平显著升高;在发生甲基化的位点中,攻毒组和对照组的mCpG占比分别为83.06%和81.91%,mCpG为最主要的甲基化形式。本研究共筛选出差异甲基化区域(DMRs) 626 677个,注释到23 706个功能基因。转录组测序共检测到29 290个基因,筛选到差异表达基因496个,其中,上调基因214个,下调基因282个。在基因组甲基化与转录组的关联分析中,筛选到180个负相关关联基因,其中,差异甲基化区域位于启动子区域的负相关基因为60个。对负相关关联基因的GO和KEGG富集分析,筛选到相关通路和LRR、hsp20和CARD等关键基因。本研究将为解析刺参抗病的表观遗传调控机制提供数据,也为刺参抗病性状选育提供科学参考。     

圆斑星鲽sox9基因的克隆与表达

本研究筛选到圆斑星鲽(Verasper variegatus)的性别相关基因sox9,并通过RACE技术获得了全长序列,基因全长为3287 bp,包括1431 bp的ORF,编码477个氨基酸,368 bp的5¢ UTR和1488 bp的3¢ UTR。在3¢ UTR中有多聚腺苷酸尾和加尾信号AATAAA。通过荧光定量PCR测定了sox9基因在圆斑星鲽成鱼不同组织中的表达水平,发现sox9基因在圆斑星鲽的脑、眼、鳃、心、肝、胆、肠、精巢、卵巢、肾和肌肉等各个组织中都有不同程度的表达。在鳃、脑和精巢组织中检测到较高水平的sox9转录,其中精巢中的转录水平显著高于其他组织,sox9基因在性腺中的表达显示出性别两相性差异。其在精巢中的表达水平要显著高于卵巢,说明sox9基因与雄性性腺发育相关。通过测定sox9基因在圆斑星鲽幼鱼不同发育时期(20、30、40、50、60、70和80日龄)的表达水平,发现其在20~50日龄表达量逐渐下降,在60日龄时表达量上升,推测表达量上升可能与幼鱼性腺分化相关。     

17β-雌二醇对牡蛎性腺发育中P450基因表达的影响

利用DD RT-PCR技术筛选出牡蛎中受雌激素调节的基因并对其进行测序,得到5条有注释的基因。采用RACE的方法获得了P450基因1400bp的cDNA序列,其中包含一个975bp的开放阅读框。序列比对表明,该基因属于CYP4V2家族。系统进化树分析表明所获得的P450序列与袖扣海兔螺的CYP4V1和CYP4V2并到一起,与其他无脊椎动物的P450基因具有共同起源,但与脊椎动物的P450基因起源不同。应用Real-time PCR技术检测雌、雄长牡蛎在雌激素刺激后不同时间段P450的转录水平的变化。结果表明在17β-雌二醇刺激后,雌性牡蛎处理组性腺组织P450表现出具有显著的时间依赖性,在性腺被刺激后5d和10d时,P450基因的表达量达到最高,而雌激素对雄性牡蛎性腺组织P450的表达无显著影响。雌激素可能通过对P450的表达正反馈调控影响雌性牡蛎的性腺发育。     

不同倍性鲫鲤MeCP2基因分子克隆及时空表达分析

为了探寻不同倍性鱼生殖育性的表观遗传调控规律,实验基于不同倍性鲫鲤精巢的转录本信息库设计引物克隆了不同倍性鲫鲤的MeCP2基因。序列比对结果发现,在远缘杂交的过程中基因组发生了遗传重组和变异。基于序列公共部分设计引物,通过半定量PCR和实时定量PCR的方法分析了MeCP2基因在不同倍性鱼发育过程中的时空表达特征。结果还发现,MeCP2基因在所有组织中都有表达,但在脑组织中的表达量最高;纵向对比性腺的发育过程,MeCP2基因的表达量会随着性腺发育成熟而下降;横向对比不同倍性鱼的性腺发育,MeCP2基因在三倍体鱼卵巢中的表达量明显高于二倍体和四倍体鱼,但在精巢中的表达量随鱼的倍性增加而增加。研究表明,MeCP2基因的表达与不同倍性鱼的卵巢发育密切相关,这为研究鱼类生殖不育的分子机理及其在水产育种上的应用奠定了坚实的基础。     

仿刺参纤维胶凝蛋白基因的分子特征及表达分析

纤维胶凝蛋白是非特异性免疫系统中的重要分子。通过转录组测序及cDNA末端快速克隆技术得到一条仿刺参纤维胶凝蛋白基因的全长cDNA序列,并将其编码的蛋白命名为仿刺参纤维胶凝蛋白-1。获得的基因cDNA全长为1951bp,其中5′-末端非翻译区为397bp,3′-末端非翻译区为666bp,开放阅读框为888bp,编码295个氨基酸,N端16个氨基酸为信号肽,信号肽后面有两个G-X-Y重复序列,C端为纤维蛋白素原结构域。采用实时荧光定量PCR方法分析了仿刺参纤维胶凝蛋白-1基因在仿刺参幼参不同组织及细菌脂多糖刺激后的时序表达规律,结果显示仿刺参纤维胶凝蛋白-1基因在仿刺参的肠道、呼吸树、体腔细胞和体壁均有表达,且肠道的表达量最高;脂多糖刺激后,4种组织的仿刺参纤维胶凝蛋白-1基因表达量均有变化,且以肠道和体壁表达量的变化最为显著;此外,仿刺参纤维胶凝蛋白-1基因在仿刺参4种组织中的表达量变化具有不同的时序性,表明仿刺参纤维胶凝蛋白-1可能在仿刺参免疫应答中起重要作用。     

牙鲆dazl基因的克隆与表达分析

dazl(deleted in azoospermia-like)基因是多种物种精子发生的重要调控因子,为探讨该基因在牙鲆(Paralichthys olivaceus)性腺发育分化中的作用,本研究采用同源克隆和RACE技术克隆了牙鲆dazl基因的cDNA序列,利用生物信息学方法分析了其序列结构,并利用实时荧光定量PCR技术检测了该基因在牙鲆成体组织和早期发育阶段的表达情况。结果发现:牙鲆dazl cDNA序列全长2 031 bp,包括105 bp的5'端非翻译区,1 275 bp的3'端非翻译区和编码217个氨基酸残基的651 bp开放阅读框;氨基酸同源序列比对显示该序列存在一个高度保守的RRM(RNA recognition motif)结构域,且该结构域在鱼类中具有100%的序列一致性;荧光定量PCR结果显示dazl基因主要在牙鲆性腺中表达,且在精巢中的表达高于卵巢;在牙鲆早期发育阶段,dazl基因在未受精卵、受精卵及囊胚期均有较高的转录本,而在原肠胚期表达开始降低,至孵化后3 d一直保持较低的水平,这些丰富表达的母源性dazl mRNA可能参与了胚胎发育中原始生殖细胞的形成。本研究为进一步阐明dazl基因在牙鲆生殖发育中的功能奠定了基础。     

Caspase-3在黄鳝性腺性逆转过程中的表达及定位分析

为探究黄鳝( Monopterus albus )Caspase-3在性腺性逆转发育过程中的功能和作用,实验扩增黄鳝 caspase-3 的部分序列,检测了其在不同发育时期性腺中mRNA表达水平、蛋白相对含量以及表达位置。通过PCR扩增获得黄鳝 caspase-3 基因cDNA序列,推导的氨基酸序列对比发现,黄鳝 caspase-3 与大黄鱼( Larimichthys crocea )和鳜( Siniperca chuatsi )亲缘关系最近。实时定量PCR ( qRT-PCR)结果显示, caspase-3 mRNA在黄鳝各发育时期性腺中均有表达,在卵黄生成期性腺中表达量最高,并伴随性腺向雄性发育表达量呈下降的趋势;而其蛋白相对含量在性腺性逆转过程中呈现逐渐上升的趋势,间性晚期最高。免疫组织化学染色结果显示,Caspase-3蛋白阳性信号定位于卵黄生成期卵母细胞细胞质、皮质小泡期卵母细胞的细胞核和颗粒细胞,以及各个发育时期性腺中初级生长期卵母细胞的细胞质和细胞核。综上,Caspase-3与黄鳝性逆转过程关系密切,推测其可能参与了性逆转过程中卵母细胞的凋亡过程。     

An overview of functional and stereological evaluation of spermatogenesis and germ cell transplantation in fish

Although there are almost thirty-thousand species of fish living in a great variety of habitats and utilizing vast reproductive strategies, our knowledge of morphofunctional and quantitative aspects of testis structure and spermatogenesis is still incipient for this group of vertebrates. In this review, we discuss aspects that are important to better understanding of testis structure and function, and of the development of germ cells (GC) during spermatogenesis. To achieve this, we have recently completed a number of studies presenting morphometric and functional data related to the numbers of GC and Sertoli cells (SC) per each type of spermatogenic cyst, the number of spermatogonial generations, the SC efficiency, and the magnitude of GC loss that normally occurs during spermatogenesis. We also investigated SC proliferation and the relationship of this important event to early spermatogenic cysts. The available data strongly suggest that SC proliferation in sexually mature tilapia is the primary factor responsible for the increase in testis size and for determination of the magnitude of sperm production. The influence of temperature on the duration of spermatogenesis in tilapia was also evaluated and we have used this knowledge to deplete endogenous spermatogenesis in this teleost, in order to develop an experimental system for GC transplantation. This exciting technique results in new possibilities for investigation of spermatogenesis and spermatogonial stem cell biology, creating also an entirely new and promising scenario in biotechnology—transgenic animal production and the preservation of the genetic stocks of valuable animals or endangered species.     

海州湾大竹蛏种质资源保护区生态环境质量分析与评价

本研究根据2018年5、8月对海州湾大竹蛏(Solen grandis)国家级水产种质资源保护区表层海水营养盐、重金属、石油类及相关环境因子的调查数据,运用综合质量指数法对该保护区海水生态环境质量进行综合评价,并采用富营养化指数和有机污染指数从不同角度分析该保护区的海水环境质量。结果显示,除部分站位无机氮(DIN)含量略超标外,海州湾保护区其他监测因子均符合国家Ⅰ类海水水质标准。2018年海州湾保护区海水综合质量指数范围为0.31~0.39,属于尚清洁等级。N/P值均大于Redfield系数,且磷酸盐(PO4-P)含量较低,表明该海域营养盐结构为磷限制。富营养化指数范围为0.05~0.27,海水为贫营养状况。有机污染指数范围为-0.24~0.81,该水域有机污染程度较轻。与2008~2013年历史调查数据相比,近年来,海州湾保护区海水生态环境质量保持较好。     

Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean

The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna.     

Growth performance,digestive enzyme activity and immune response of Japanese sea bass,Lateolabrax japonicus fed with fructooligosaccharide

In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg.     

Involvement of gonadal steroids in final oocyte maturation of white perch (Morone americana) and white bass (M. chrysops): in vivo and in vitro studies

Plasma estradiol-17 (E₂), testosterone (T), 17,20-dihydroxy-4-pregnen-3-one (DHP) and 17,20,21-tri-hydroxy-4-pregnen-3-one (20-S) levels were measured by radioimmunoassay (RIA) in white perch (Morone americana) and white bass (M. chrysops) that were induced to undergo final oocyte maturation (FOM) with human chorionic gonadotropin (hCG). Plasma DHP levels increased in females of both species in association with oocyte germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) and decreased thereafter. Plasma 20-S levels also increased with oocyte GVM in white bass, but were several-fold lower than DHP levels. Circulating E₂ and T levels were greatest during GVM and GVBD in both species and decreased to low levels during oocyte hydration and ovulation. Follicles from white perch and white bass which received a priming injection of hCG in vivo, produced both DHP and 20-S in vitro after exposure to hCG and their oocytes underwent GVBD. Ovarian incubates from unprimed fish of either species produced only E₂ and T and their oocytes did not complete GVBD. Oocytes from unprimed bass, but not perch, matured when follicles were exposed to hCG in vitro. Both trilostane and cycloheximide blocked in vitro production of DHP and 20-S and oocyte GVBD by white perch follices. DHP and 20-S were equipotent inducers of FOM in the GVBD bioassay. None of several other structurally-related steroids tested were effective within a physiological range of concentrations. These results indicate a role for DHP and 20-S in the control of FOM in white perch and white bass.     

Cardiac,ventilatory and metabolic responses of two ecologically dissimilar species of fish to waterborne cyanide

Changes in heart rate, ventilatory activity and oxygen consumption were determined in trout (Salmo gairdneri) and brown bullhead catfish (Ictalurus nebulosus) during exposure to a steadily increasing concentration of waterborne cyanide selected to produce death in 8–9 hours for each species. The lethal cyanide concentration for the bullheads was an order of magnitude higher than for trout. Trout developed an immediate and gradually increasing bradycardia throughout the exposure period. Cyanide produced tachycardia in the bullhead followed by a gradual onset of bradycardia as the concentration of cyanide was raised. Pericardial injection of atropine (a muscarinic cholinergic antagonist) indicated that bradycardia in the trout was due initially to increased vagal tone but later due to the direct effect of cyanide on the heart. Hyperventilation in the trout persisted throughout the exposure period, although the rate and amplitude fluctuated and was variable between individual fish. During the last hour of exposure (highest cyanide concentration), ventilation was characterized by rapid, shallow breaths followed by a sudden respiratory arrest. The bullheads exhibited hyperventilation during the first 3 hours of exposure followed by a gradual, linear drop in ventilation rate and amplitude until death occurred. Cardiac and ventilatory responses in both species were attributed to stimulation of central and peripheral chemoreceptors by cyanide. Evidence is presented which suggests the initial response in the bullheads was due, at least in part, to gustatory stimulation by the cyanide. Oxygen consumption of the trout remained above pre-exposure levels for the majority of the test period. Oxygen consumption in the bullhead paralleled the changes in heart and ventilatory rates. Whole-body lactate levels of fingerlings of both species during cyanide exposure were measured to estimate the extent of anaerobiosis. Whole-body lactate levels were much greater in the bullheads than the trout, indicating a higher capacity for anaerobiosis, possibly due to a greater fuel supply. Overall, the trout responded to cyanide in a manner similar to that produced by environmental hypoxia whereas the bullheads experienced a gustatory stimulus which masked the hypoxia-like response.     

An overview of stress physiology of fish transport: changes in water quality as a function of transport duration

This study brings an integrated analysis about the relationship between water deterioration and its physiological consequences in live fish transport. The analysis was focused on the transport water and its deterioration, and physiological challenges imposed on the fish. Usual commercial handling procedures employed to mitigate fish stress during transport were discussed. Future topics of research for the establishment of safer fish transport protocols were proposed. Transport was classified into short (≤8 h) or long transport (>8 h). The main issue in short transports should be the prevention of water pH reduction, while in long transports it is the increase in ammonia. Plasma cortisol is the most employed marker for stress and is acutely elevated upon short episodes of transport, but remains elevated even in long‐transport events. Plasma glucose is perhaps a better marker for handling stress. Plasma lactate, pH, osmolality CO₂ and ions should be more often evaluated. Plasma Na⁺ and Cl^‐ are very useful markers of acidosis, due to their respective exchange for H⁺ and , for acid–base regulation. The establishment of species‐specific transport protocols should be preceded by such combined analyses of water and physiological parameters.