Cd2+对萼花臂尾轮虫的毒性研究

Cd2 对萼花臂尾轮虫24 h和48 h的LC50分别是1.488 mg/L和0.526 mg/L.在Cd2 存在的条件下,其对萼花臂尾轮虫的存活率、繁殖率、净生殖率和内禀增长率有显著的抑制作用,混交百分率和休眠卵数量则明显增加.     

氟他胺对萼花臂尾轮虫的毒性影响

采用两天种群增长和生命表,研究了不同浓度氟他胺对萼花臂尾轮虫(Brachionus calyciflorus)的毒性影响。结果显示:轮虫种群增长率与氟他胺浓度对数负相关。氟他胺对萼花臂尾轮虫种群增长率的半效应浓度(EC50)、最低可见效应浓度(LOEC)和无可见效应浓度(NOEC)分别为4.31、3.17和1.59 mg/L。氟他胺对萼花臂尾轮虫的内禀增长率和世代时间有显著的影响(P<0.05);内禀增长率随氟他胺浓度升高而下降,其中1.59 mg/L和3.17 mg/L氟他胺浓度组内禀增长率分别比对照组降低了8.98%和17.95%(P<0.05),其无效应浓度为0.8 mg/L;而3.17 mg/L浓度组的世代时间显著延长(P<0.05)。实验结果表明内禀增长率是检测氟他胺对萼花臂尾轮虫毒性最敏感的指标。     

几种轮虫需精卵休眠时间的初步研究↑（*）

本文报道了角突臂尾轮虫，萼花臂尾轮虫，褶皱臂尾轮虫部分新产需精卵的自然休眠期分别为９天，６－７天，大于１２天，晶囊轮虫部分需精卵的休眠显８－１１ｈ。光照是轮虫新产需精卵萌发的必要条件。     

(鱼央)属4种鱼SRY、SOX和HOX基因同源序列的PCR扩增分析

选用根据人类SRY基因HMG保守区序列、HMG-box序列以及HOX基因保守序列设计的3对引物,分别对白缘(Liobagrus marginatus)、大拟缘(Liobagrus marginatoidesW u(b ig))、小拟缘(Liobagrus margina-toidesW u(sm all))、黑尾(Liobagrus nigricauda)4种鱼的雌雄基因组进行了PCR扩增。结果在4种鱼的雌雄基因组中发现1个1 200 bp左右的SRY基因同源序列片段,3个分别为200 bp、500 bp和1 200 bp左右的SOX基因同源序列片段,2个分别为150 bp和200 bp左右的HOX基因同源序列片段。对结果进行分析显示:SRY、SOX和HOX基因同源序列在4种雌雄个体间和种间无性别特异性和种属特异性,并且在SRY和SOX基因同源序列中可能含有内含子。     

萼花臂尾轮虫室内规模培养技术

萼花臂尾轮虫具有生长快、繁殖力强、营养丰富等特点,是水产苗种开食和早期培育阶段的优良饵料,可以在淡水中培养。结合轮虫室内高密度培养特点及自身实践经验,对萼花臂尾轮虫室内规模培养技术作简要总结。     

萼花臂尾轮虫的培养及营养强化技术

轮虫是浮游生物的重要组成部分,广泛分布于淡水、半咸水和海水水域中。萼花臂尾轮虫隶属臂尾轮虫科、臂尾轮虫属,为淡水轮虫,俗称灰水,系池塘、湖泊、江河中常见的浮游动物,     

基于ND4和ND5基因序列研究八种臂尾轮虫的系统关系和分类地位

通过对尾突臂尾轮虫、裂足臂尾轮虫、剪形臂尾轮虫、方形臂尾轮虫、红臂尾轮虫、镰形臂尾轮虫、矩形臂尾轮虫和十指臂尾轮虫8种臂尾轮虫的ND4和ND5基因序列进行扩增、测序,结合Gen Bank数据库中的褶皱臂尾轮虫、萼花臂尾轮虫、转轮虫和旋轮虫的ND4和ND5基因序列,使用MEGA6.0软件构建这12种轮虫系统发生树(NJ树、ME树、UPGMA树),探讨了8种臂尾轮虫之间的系统关系。结果显示,本研究所涉及的轮虫ND4和ND5基因序列差异百分比均值为40.3%,可作为分子标记应用于轮虫属内种间系统关系研究;系统树所显示亲缘关系与形态学研究基本一致,均支持将十指臂尾轮虫、裂足臂尾轮虫归入臂尾轮属。     

暗纹东方鲀仔鱼的生长特性及其开口饵料的研究

用计算机图形分析技术，通过计算机软什系统对暗纹东方鲀仔鱼的生长情况进行了周长、面积和长度3个参数的数学统计，求得其经验公式。研究了萼花臂尾轮虫、草履虫和蛋黄颗粒3种不同饵料对暗纹东方鲀仔鱼的摄食和生长的影响。结果表明：6日龄前是卵黄囊期营内源性营养，5～13日龄是混合性营养期，也是决定仔鱼成活率的关键期。喂以萼花臂尾轮虫实验组的仔鱼具有最高的存活率和最大的生长率。因此，萼花臂尾轮虫是暗纹东方鲀仔鱼最适宜的开口饵料。     

黄鳝ghrelin基因的克隆及分子特征分析

Ghrelin是联系生殖和能量代谢的重要桥梁信号分子,通过克隆黄鳝(Monopterus albus)的ghrelin基因并对其基因结构和功能进行了初步分析;运用c DNA末端快速扩增技术获得了黄鳝ghrelin基因的c DNA序列和DNA序列全长。结果表明,黄鳝ghrelin基因c DNA全长552 bp(Gen Bank accession no.JX122807),包括115 bp的5'端非编码区、324 bp的完整开放阅读框以及113 bp的3'端非编码区;DNA序列全长1323 bp,由3个内含子和4个外显子构成,内含子剪切位点具有典型识别核苷酸GT/AG,3个内含子分别为594 bp、84 bp和93 bp,4个外显子长度分别为229 bp、78 bp、112 bp和133 bp。氨基酸序列分析显示,ghrelin基因推导的Ghrelin蛋白前体原(propreghrelin)序列由26 aa的信号肽、19 aa的成熟肽以及C端氨基酸残基等构成;其中,成熟肽第3位为丝氨酸(Ser3),是Ghrelin的酰基化位点;C端氨基酸残基序列极可能包括与Ghrelin成熟肽功能相互拮抗的肥胖抑制素(Obestatin)。氨基酸的同源性及进化关系分析表明,黄鳝与某些鲈形目鱼类的蛋白前体原存在高度相似性,且在进化上黄鳝与较高级的鲈形目、鲽形目鱼类聚为一支。ghrelin基因结构及其蛋白质某些氨基酸残基序列的高度保守,预示着Ghrelin在脊椎动物中有着重要的生理功能与类似的作用机制。     

两株蛋白核小球藻 rbcS cDNA 全序列的克隆和分析

以2株蛋白核小球藻(Chlorella pyrenoidosa)F-9和820为实验材料,根据已知绿藻rbcS基因设计简并引物,分别克隆到2条245bp的cDNA片段,以此片段为基础使用cDNA末端快速扩增(RACE)技术,获得了2条长度分别为861bp和907bp的rbcS基因。序列分析表明,蛋白核小球藻F-9和820的rbcS分别编码181和184个氨基酸,编码区具有与高等植物rbcS保守序列YYDGRYWTMWKLPMFG相类似的结构,起始密码子附近有Kozak保守序列(A/GXXATGG),3′非翻译区有加尾信号TGTAA。同源性分析结果表明,F-9与蛋白核小球藻(GenBank登录号BAE48226)的相似性最高(91%),而F-9与820、F-9和普通小球藻(C.vulgaris)的相似性分别为64%和50%。这2条rbcS基因与其他绿藻和高等植物也表现了广泛的同源性,但相似性不高。该研究旨在为rbcS基因的功能和表达研究奠定基础。     

Molecular analysis of grass carp (<Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>) by SRAP and SCAR molecular markers

The sequence-related amplified polymorphism (SRAP) technique was used to analyze the gene differentiation between two cultured populations [Freshwater Fisheries Research Center (FFRC) and Qianzhou populations] and one wild population (Hanjian population) of grass carp (Ctenopharyngodon idella). Some loci showed quite different genetic frequencies, attributable to artificial selection, which imply that these fragments are putative markers of germplasm identification. We developed a simple and effective method to further characterize these SRAP fragments. Specific SRAP bands were cut directly from polyacrylamide gels, re-amplified, cloned, and sequenced. Twenty-one putative genetic markers were sequenced, ranging from 137 to 357 bp. The sequences were submitted to the database of the Genome Sequence Survey. A BLAST analysis showed that eight SRAP fragments were highly similar to functional genes, whereas the other 13 had no similarity, indicating that these markers are tightly linked to the germ identification trait although only eight are functional genes. Three primers were designed according to this sequence information and used for PCR amplification of the three populations. A sequence-characterized amplified region (SCAR1) was positively amplified in the artificially cultured populations but not in the wild population. The frequency of the SCAR3 marker in the cultured populations was 87% (26/174), whereas it was only 6% (6/100) in the wild population. A specific band was isolated from all individuals in the wild population with the SCAR3 primers, whereas the specific band was amplified from only seven individuals in the FFRC population and from none of the Qianzhou population. The frequency of SCAR2 in the artificially cultured populations was 96.5%. These results indicate that SCAR1 could be used as a specific molecular marker for population identification. The SCAR markers used in this study offer a powerful, easy, and rapid method for genetic analysis and the discrimination of different populations.     

大西洋鳕微卫星引物对太平洋鳕的适用性

利用Genbank中大西洋鳕的7对微卫星引物,对太平洋鳕基因组DNA进行PCR扩增。这7对引物均可较稳定地扩增出条带,它们分别是Gmo2、Gmo3、Gmo8、Gmo19、Gmo35、Gmo36、Gmo37,并且扩增出的7个位点均表现出多态性,共获得69个等位基因,每个位点的等位基因数目为4～17,大小为111～227bp。在7个位点上2种鱼的等位基因的大小存在着不同程度的差异。其中Gmo37在大西洋鳕和太平洋鳕中扩增出的条带的长度差异最大,基本无重合部分,分别为220～290bp和144～226bp,本研究结果以期对2种鳕科鱼类遗传结构的进一步研究提供帮助。     

条斑紫菜6个品系的SRAP分析

为鉴别条斑紫菜不同品系的种质,使用相关序列扩增多态性(sequence-related amplified polymorphism,SRAP)标记对条斑紫菜的5个选育品系和1个野生品系进行遗传分析,结果从35对引物组合中筛选出可扩增出稳定清晰条带的组合11对,共获得131个扩增位点,其中多态性位点125个,多态性比例高达95.42%。6个品系 间的遗传距离为0.364 3～0.867 9,平均为0.593 0。用UPGMA法进行聚类分析,结果将6个品系分为2个群,所反映的亲缘关系与各品系的来源基本一致,说明SRAP 标记技术可以成为条斑紫菜品系间遗传分析的有效工具。在131个多态性位点中,选择扩增出的4个位点构建了6个品系的指纹图谱。另外,通过ME1/EM6引物组合 扩增得到耐高温品系TM-18的特异性条带,经回收测序和重新设计引物,该条带在其丝状体和叶状体DNA中均能稳定地被扩增出来,可用于该品系的种质鉴别 。     

栉孔扇贝sox9基因的cDNA克隆及其在不同发育时期性腺中的表达特征

SOX9是SOX家族的SOXE亚族成员,在脊椎动物骨骼发育、胰腺发育、性别决定与分化以及肿瘤形成中发挥重要的作用。sox9在不同种类脊椎动物性腺中的表达存在差异,在无脊椎动物性腺中的表达特征尚不清楚。本研究利用RACE技术克隆了栉孔扇贝(Chlamys farreri)sox9(Cf-sox9)全长的c DNA序列,其长度为2835 bp,开放阅读框为1413 bp,编码470个氨基酸。预测的氨基酸序列具有SOX家族的HMG-box和SOXE亚族的高保守区域,但没有脊椎动物羧基端的Pro-Gln-Ser rich区域。原位杂交和免疫组织化学技术确定Cf-sox9 m RNA和Cf-SOX9蛋白均定位在栉孔扇贝精巢和卵巢的所有生殖细胞中,并且在不同发育时期性腺中呈现了类似的表达规律,即在精巢中,阳性信号在精母细胞中最强,在精子中最弱;在卵巢中,阳性信号在卵原细胞、卵母细胞以及成熟卵中呈现逐渐减弱的趋势。这一表达特征与脊椎动物性腺中多样的性别差异表达特征不同,提示sox9在贝类性腺发育和配子发生中的作用可能与大多数脊椎动物不同。     

热应激对剑尾鱼HSP70家族两成员基因表达的影响

采用RT-PCR方法扩增实验动物剑尾鱼（Xiphopohorus helleri）纯系RR-B的HSP70家族两个成员的eDNA片段，并将其克隆到PMD18-T载体中测序，将测序结果与GenBank中的核苷酸序列和推导的氨基酸序列进行同源性比较。同时利用RT-PCR半定量方法研究热应激时两个成员在剑尾鱼组织中的表达。通过克隆获得了剑尾鱼的HSP70家族两个新成员的cDNA片段，两序列均包含HSP70家族的特征性签名序列和热休克蛋白的定位序列；通过对克隆片段与已发表的青锵（Oryzias latipes）等鱼类HSP70的核苷酸序列和其编码的氨基酸序列同源性比较，发现核苷酸序列同源性较高，成员一和成员二分别为85％-89％和82％-99％；氨基酸序列同源性更高，成员一和成员二分别为97％-99％和93％-99％。一般条件下，两成员在剑尾鱼肝脏、脾脏、肾脏和心脏中不表达，但热应激能刺激成员一在剑尾鱼脾脏中表达，成员二在肝脏、脾脏、肾脏和心脏中强烈表达，并可能在参与热应激保护方面起更大作用。     

对虾抗病性状遗传标记的RAPD分析

对人工选育的第3代中国对虾和选育的第1代凡纳对虾进行个体人工感染WSSV后，选取感染WSSV十余天但仍健康的对虾为实验材料，用220个随机引物进行RAPD分析，得到抗病性状相关的特异性遗传标记99个。其中中国对虾抗病组特异性片段出现了18个，片段大小在460－2305bp之间；凡纳对虾抗病组特异性片段产生81个，片段大小在435－2287bp。77个引物在两种对虾的抗病组扩增出特异性遗传标记，其中有4个引物各获得了三个特异性片段，有13个引物各获得了两个特异性片段，其余61个引物各获得了1个特异性片段。     

Sequence characterized amplified regions (SCAR) reveal candidate markers linked to growth traits in the Chinese shrimp Fenneropenaeus chinensis

Growth traits in 150 Chinese shrimps Fenneropenaeus chinensis were analyzed by the sequence characterized amplified regions (SCAR) technique in two populations of sixth-generation cultured shrimps with different ranges of body lengths (CP-a and CP-b) and in the wild-type population (WP). First, 240 random primers were used to screen polymorphic fragments by random amplified polymorphic DNA (RAPD). Population analysis revealed nine RAPD markers correlated with growth traits, including seven positively and two negatively correlated ones. The sequences of the RAPD markers were then used to design longer primers for SCAR. Six primer pairs were obtained, and two of these produced polymorphic fragments among the three groups. One amplified 39, 26 and 27 polymorphic fragments, with band frequencies of 78, 52 and 54% in the CP-a, CP-b and WP groups, respectively. The distributions of polymorphic fragments in the three groups were significantly different (P < 0.05) according to a χ ² test, indicating that they may be candidate markers linked to growth traits. Another primer pair amplified three alleles, resulting in six combinations of genotypes among three groups. Since allele A₁ was only found in the population of shorter shrimps, it may be a negative growth marker.     

浅色黄姑鱼线粒体16S rRNA基因片段序列特征分析

PCR扩增100个浅色黄姑鱼个体的线粒体16SrRNA基因片段序列,得到大约620bp的扩增产物。将其中5个个体的扩增产物进行测序和同源比对,得到468bp可供比对分析的片段。比对结果表明,5条序列包括两个单倍型,两个单倍型之间有1个碱基突变。PCR-RFLP分析结果显示,两个种群的100个样品中98%的个体为其中一种单倍型,只有2%的个体呈另一种单倍型,表明这两个种群的遗传多样性较低。两个单倍型平均碱基组成为:T22.0%,C26.3%,A29.8%,G21.9%,GC含量平均为48.2%。与GenBank中石首鱼科7属9种的11条同源序列比对,得到429个比对位点,其中包括69个简约信息位点、55个单突变子和16个插入/缺失位点。聚类分析显示,浅色黄姑鱼与黄姑鱼亲缘关系较近,与形态分类相符。     

Application of RAPD and cytochrome b sequences to the study of genetic variations in F1 and F2 generations of two different strains of guppy (Poecilia reticulata, Peter 1854)

The DNA of two laboratory strains of guppy (Poecilia reticulata), Black Yellow (BYS) and Red Flame (RFS), was studied with respect to their colour differences, in two generations (F₁ and F₂). Their varying morphological colours were related to their cloned fragments of cytochrome b mitochondrial DNA (mtDNA) and random‐amplified polymorphic DNA‐polymerase chain reaction (RAPD‐PCR). The F₁ generation was characterized by various forms. The male BYS could be divided into 68% having black–yellow tails and 14% having black–yellow–red tails. The other variations were found in very low percentages. The percentage of black–yellow‐tailed males increased to 85 in the F₂ generation, and the percentages of the various other forms consequently decreased. Only 63% of BYS males had black–yellow dorsal fins in the F₁ generation, but this percentage increased to 84 in F₂. Compared with the males, fewer variations were found in female colour patterns in the F₁ generation. A high percentage of BYS females (81%) colour was found with no significant increase in the F₂ generation. However, variations decreased in the F₂ females. On the other hand, a very high variation was found in female fins in the F₁ generation: only 32% were of BYS colour and 25% had no‐colour fins. However, a significant increase in BYS colour was found in the F₂ generation (61%) and 39% had no colour. The variation in RFS was lower than BYS in the F₁ generation: 81% of the F₁ males had red–yellow tails with colour, 46% of the fins were yellow and 36% were red–white. In females, a very high percentage (84%) had red–yellow tails and 76% had no‐colour dorsal fins. Mitochondria DNA markers and genomic DNA were studied in various laboratory strains. In the clone of the fragments of cytochrome b, the bands correlated to the colour phenotype. A fragment of the cDNA sequence was determined from a 268‐bp cloned with fragments of the guppy cytochrome b mtDNA gene. The genes varied for the two strains in only two base pairs, starting at the nucleotide position 171 and ending at position 174. Three primers showed good results in the RAPD‐PCR and were found suitable for the study of DNA variations in guppy. The high variation detected in BYS, in comparison with RFS, was reflected by changes in band‐sharing (BS) values ranging from 0.66 to 1, versus 0.8 to 1 in RFS.