草鱼肠系膜脂肪组织高质量总RNA提取方法的研究

基于传统的酸性酚—异硫氰酸胍—氯仿一步提取法,比较分析多种优化操作步骤,摸索出一种高质量提取体质量为80~150 g草鱼肠系膜脂肪组织总RNA的改良方法。试验结果显示,相较于肝脏、脾脏、肠道等脏器组织,草鱼肠系膜脂肪组织RNA丰度低,且极易在样品前处理阶段出现顽固性降解问题。探索发现,将取样量增至约30 mg,可提升RNA产量以满足常规试验需求。针对降解难题,改良常规的样品前处理技术流程,采用鲜样液氮速冻,冻样直接放入TRIzol试剂中裂解,并即刻进行长时间机械匀浆,时长约3 min等核心操作步骤,可显著降低脂肪组织样品RNA的降解。Agilent生物分析仪检测结果显示,改良方法提取的草鱼肠系膜脂肪组织RNA完整度高,关键RIN值为8.7~9.0。研究推测,长时间机械匀浆所形成的持续剪切冲击力或许有助于TRIzol试剂中的异硫氰酸胍等成分突破油滴阻碍而有效抑制内源性RNA酶。本方法提取的草鱼脂肪组织总RNA质量可满足高通量转录组测序要求。     

Use of Microsatellite DNA Profiling to Identify Japanese Flounder,Paralichthys olivaceus of Hatchery Origin

Japanese flounder, Paralichthys olivaceus, is an economically important marine fish species in Asia. A suite of 18 microsatellite markers chosen from published genetic linkage maps was used to carry out parentage assignments of 188 hatchery‐reared juveniles from a small number of breeders. The probabilities of exclusion for the 18 microsatellite markers were 0.604–0.913, and the effectiveness of combined probability of exclusion reached 100% when using the eight microsatellite markers with higher Excl 1 probabilities. The cultured and wild stocks (WSs) were differentiated in a release‐recapture population based on these markers. Of the 321 recaptured offspring, 28.34% were assigned to their parental pairs in our broodstock, whereas the remaining offspring could not be traced back to a possible sire or dam. Significant reduction in genetic diversity of the cultured stock (CS) had not been found compared with that of the WS. The results suggest that CSs released into the wild will not adversely affect the genetic structure of natural populations. Our results demonstrate that these markers provide an efficient tool for parentage assignments and genetic analysis of Japanese flounder.     

胰α-淀粉酶基因5''端调控序列与鱼类食性的关系

为了研究不同鱼类胰α-淀粉酶基因5'端调控序列转录因子差异与鱼类食性分化之间的关系。通过PCR克隆测序和查询NCBI数据库,获得32种鱼类胰α淀粉酶基因5'端824 bp序列,并对鱼类胰α淀粉酶基因5'端序列进行转录因子和系统发育分析。按不同的营养类型将鱼类分为杂食性、植食性和肉食性,通过百分比相似性分析不同食性鱼类的胰α淀粉酶基因转录因子的组成差异以及转录因子与鱼类食性的关系。结果显示:不同食性鱼类的胰α-淀粉酶基因5'端序列存在转录因子种类差异,植食性-肉食性鱼类差异主要体现在E47、C/EBPalpha、NF-Y和Pax-2,植食性-杂食性差异主要体现在deltaEF1、MyoD、NF-Y、AREB6和Pax-2,杂食性-肉食性差异主要体现在GATA-1、SRY、MyoD、HFH-8、AREB6、Pax-2、STAT5A和AP-1。系统发育结果与传统形态分类学大体相符,相同食性的鱼类并没有聚为一类。鱼类胰α-淀粉酶基因5'端调控序列中与食性分化相关的转录因子有E47、C/EBPalpha、NF-Y、Pax-2、deltaEF1、MyoD、AREB6、GATA-1、SRY、HFH-8、STAT5A和AP-1;胰α-淀粉酶基因5'端序列的转录因子与鱼类食性分化具有一定的关系。     

驯食配合饲料的大口黑鲈3个选育世代的遗传多样性分析

为了检测驯食配合饲料的大口黑鲈(Micropterus salmoides)3个选育世代群体遗传多样性水平变化,利用微卫星标记技术对驯食配合饲料大口黑鲈选育基础群体(Sp0)和第二、三和四代选育群体(Sp2、Sp3和Sp4)共240尾样品进行检测。结果显示,18个微卫星位点共获得44个等位基因。Sp0、Sp2、Sp3和Sp4的平均观测杂合度(H_o)分别为0.4895、0.4802、0.4579和0.4206,平均期望杂合度(H_e)分别为0.4615、0.4454、0.4621和0.3916,平均多态信息含量(PIC)分别为0.3791、0.3659、0.3764和0.3257。4个群体间的配对比较群体间遗传分化指数(F_(st))值在0.01612～0.16162之间、遗传距离(D_a)在0.0249～0.1434之间。遗传变异来源(AMOVA)分析显示,只有8.38%的变异来自于群体间,其余遗传变异均来自于个体间。研究表明,经连续多代选育之后,易驯食配合饲料的快长大口黑鲈选育群体具有中度遗传多样性,具备选育潜力,可继续进行选育。     

添加芽孢杆菌对草鱼池塘中真核微生物的影响

为了研究添加芽孢杆菌对池塘中真核微生物群落结构和理化因子的影响,采用高通量测序技术分析了实验组(添加芽孢杆菌池塘)与对照组(普通池塘)水体真核微生物群落结构,同时分析了两组池塘的水体理化指标。结果表明:8、9月实验组池塘水体中TN、NH_4~+-N、NO_3~--N含量显著低于对照组(P0.05)。水体中梅尼小环藻(Cyclotella meneghiniana)、红囊藻(Hedriocystis)、蓝隐藻(Chroomonas)、丝孢酵母属(Trichosporon)和小环藻属(Cyclotella)真核微生物丰度显著高于对照组(P0.05)。实验组池塘水体真核微生物Chao1指数和Shannon指数显著高于对照组(P0.05)。实验结果证实:通过向池塘添加芽孢杆菌,可以改变水体中真核微生物群落的结构,从而实现对池塘理化因子的调节。研究结果对于降低水产养殖尾水对水域环境的污染具有一定的意义。     

Whole-genome sequence of a novel Chinese cyprinid herpesvirus 3 isolate reveals the existence of a distinct European genotype in East Asia

Cyprinid herpesvirus 3 (CyHV3), also known as koi herpesvirus (KHV), can be subdivided primarily into European and Asian genotypes, which are represented by CyHV3-U or CyHV3-I and CyHV3-J, respectively. In this study, the whole genome sequence of a novel Chinese CyHV3 isolate (GZ11) was determined and annotated. CyHV3-GZ11 genome was found to contain 295,119 nucleotides with 52.9% G/C content, which is highly similar to those of published CyHV3-U, CyHV3-I, and CyHV3-J strains. With reference to CyHV3-U, CyHV3-I, and CyHV3-J, CyHV3-GZ11 was also classified into 164 open reading frames (ORF), which include eight repeated ORFs. On the basis of the 12 alloherpeviruses core genes, results from phylogenetic analysis showed that CyHV3-GZ11 had closer evolutionary relationships with CyHV3-U and CyHV3-I than with CyHV3/KHV-J, which were also supported by genome wide-based single nucleotide substitution analysis and the use of a series of developed molecular markers. This study was the first to reveal the presence of a distinct European CyHV3 genotype in East and Southeast Asia at a whole genome level, which will evoke new insights on exploring the origin, evolution, and epidemiology of the virus.     

Modeling and implementing the use of aeration to increase water temperature and dissolved oxygen in greenhouse aquaculture cages

In this study, an unique bamboo floating cage was built in order to heat the water used for rearing Nile tilapia (Oreochromis mossambicus x O. niloticus) and climbing perch (Anabas testudineus, Bloch) in Northern Thailand during the colder rainy and winter seasons. This special greenhouse fish cage (SGFC) was covered with polyethylene to heat the surface air with solar energy, and insulated to prevent heat loss. The water temperature at depths of 0.1, 0.3 and 0.8 m was modeled and measured. In various trials, the cages were aerated with and without heated air from the surface. It was found that the thermal efficiency of the SGFC integrated with hot air aeration at a water depth of 0.80 m was 32.65 %. Throughout the day, the average water temperature was 31.82 ± 0.68 °C, which was 0.76 °C higher than the water temperature outside the experiment, and the amount of oxygen in the water of 11.35 ± 0.13 mg/l. The SGFC can produce one extra fish harvest per year when compared with regular fish cages. Development equations to describe the thermal model were used for predicting the water temperature in the SGFC and thus validating the model. Moreover, the economic analysis of the SGFC found that the payback period was one year, seven months and fourteen days.     

投喂蚕豆和普通配合饲料草鱼肌肉营养成分比较分析及营养评价

对投喂蚕豆和普通配合饲料的2组草鱼（Ctenopharyngodon idellus）肌肉中的胶原蛋白、钙离子（Ca2＋）、氨基酸和脂肪酸含量进行了比较分析，并对其营养价值与食用价值进行了评价。结果显示，投喂蚕豆草鱼肌肉中胶原蛋白和Ca2＋的质量分数分别为（0．149±0．011）％和（0．036±0．011）％，比投喂普通配合饲料的草鱼分别提高了36．7％和154％。投喂蚕豆草鱼肌肉中氨基酸总量为（16．802±1．823）％，投喂普通配合饲料草鱼氨旗酸总量为（18．444±0．850）％；投喂蚕豆草鱼肌肉中4种鲜味氨基酸[天冬氨酸（Asp）、谷氮酸（Glu）、甘氯酸（Gyl）和丙氨酸（Ala）]总质量分数为（6．613±0．771）％，低于投喂普通配合饲料草鱼。投喂蚕豆荦：鱼和投喂普通配合饲料草鱼肌肉中不饱和脂肪酸（UFA）总量分别占脂肪酸总量的73．857％和76．334％，其中单不饱和脂肪酸（MUFA）分别占脂肪总量的（55．598±2．403）％和（33．832±2．755）％。结果表明，投喂蚕豆荦：鱼叽肉中胶原蛋白和Ca2＋含量比投喂普通配合饲料的草鱼有显著提高，但其鲜味程度、氨基酸以及小饱和脂肪酸含鞋比投喂普通配合饲料草鱼低。     

灰色聚类法在胶州湾富营养化评价中的应用

参照国家海水水质标准(GB3097-1997)和海水的富营养化特点,选取活性磷酸盐(PO43--P)、溶解无机氮(DIN)、溶解氧(DO)、化学需氧量(COD)和叶绿素a(Chl-a)为评价指标,将富营养化程度分为3个级别,利用灰类白化权函数描述海水富营养化的等级界限,确定各指标在灰类中的聚类权及聚类系数,确定各聚类对象的等级,在此基础上建立海水富营养化综合评价模型。以胶州湾为研究对象,对2009年5月和8月的监测数据进行分析,结果表明,胶州湾东部海域已达到富营养化,西部和北部海域处于中度营养水平,湾中和湾口附近海域处于贫营养水平,这与沿岸河流、工农业废水、生活污水和养殖废水的排放、湾内海水与外海水的交换速率以及水域水深条件密切相关。灰色聚类法评价的胶州湾水域的富营养化状态与真实情况接近,因此,利用灰色聚类法评价海水富营养化是一种快速、简便、客观的方法。     

自动采集转轮及其在鱼类资源调查中的应用

介绍了一种自动采集转轮的结构、工作原理及其在鱼类资源调查中的应用。自动采集转轮特别适合在小河流及溪流中自动采集小型鱼类,并且选择性小、活体采样、对鱼类无大的损伤、可长期自动运行、成本低廉。