三个三倍体鲫鱼品系及野鲫mtDNA的比较研究

用16种限制性内切酶研究了银鲫(3N=156～162)、彭泽鲫(3N=162)和缩骨鲫(3N=150)3个三倍体鲫鱼品系及野鲫(2N=100)的线粒体DNA。有6种酶在种系间和种系内产生限制性片段长度多态性(RFLPs),银鲫共存在4种单倍型,彭泽鲫2种,野鲫3种,缩骨鲫1种。彭泽鲫和银鲫拥有相同的常见单倍型,缩骨鲫的单倍型属于野鲫的常见型。根据限制性位点的变异数据,计算了单倍型间的相似性、核苷酸多样性、品系内核苷酸多样性和品系间的遗传距离,确定彭泽鲫属于银鲫的一个地方品系,缩骨鲫属于野鲫的一个地方品系。根据核苷酸的差异,推算出银鲫和野鲫两个亚种的分化大约在11万年前完成。     

洞庭青鲫与其他六个鲫鱼品系线粒体DNA控制区的比较分析

运用PCR扩增、克隆、测序等技术,在获得洞庭青鲫和彭泽鲫线粒体DNA控制区全序列的基础上,对洞庭青鲫、彭泽鲫、普通鲫、红鲫、白鲫、A系和D系银鲫等7个鲫鱼品系线粒体DNA控制区的碱基组成、变异情况、序列结构和系统进化进行了比较分析。结果表明,7个鲫鱼品系线粒体DNA控制区碱基的平均组成为A:(32.7±0.16)%,C:(20.4±0.37)%,G:(14.1±0.08)%,T:(32.8±0.36)%,序列分歧率为0—5.7%,其中红鲫和白鲫的分歧率最大(5.7%),彭泽鲫、A系和D系银鲫之间最小(0)。洞庭青鲫与白鲫的分歧率为5.6%,与彭泽鲫、A系和D系银鲫为2.2%,与红鲫和普通鲫分别为0.8%和0.7%。对比哺乳动物线粒体DNA控制区结构,并参照其他鱼类序列,将7个鲫鱼品系的控制区分为终止序列区、中央保守区和保守序列区三个区域。同时识别了7个鲫鱼品系中一系列保守序列,并给出了它们的一般形式。序列差异和系统进化分析表明,在7个鲫鱼品系中,洞庭青鲫与普通鲫和红鲫的亲缘关系最近,与彭泽鲫、A系和D系银鲫亲缘关系次之,与白鲫的亲缘关系最远,而彭泽鲫、A系和D系银鲫三者在线粒体DNA控制区的相似性和分歧率上则表现为是同一个鲫鱼品系。     

一种乌鳢与斑鳢线粒体PCR-RFLP鉴定方法

为了高效的鉴别乌鳢与斑鳢,采用PCR-RFLP技术,对乌鳢与斑鳢开展分子生物学鉴定方法研究。通过比对乌鳢和斑鳢线粒体全序列,发现1处单核苷酸多态性位点,可以明确区分两个物种。利用1对引物对乌鳢与斑鳢线粒体基因组该区域进行PCR扩增,用限制性内切酶EcoR I分别对扩增产物进行酶切,并用1.5%的琼脂糖凝胶检测酶切结果。PCR-RFLP检测结果显示,斑鳢的PCR扩增产物被EcoR I酶切后生成两个不同大小的片段,分别为315 bp和875 bp,乌鳢则保持不变。由此可将乌鳢与斑鳢在酶切图谱上鉴别出来。     

鲫鱼遗传多样性的初步研究

用１７种限制性内切酶对鲫属普通鲫鱼低背型,高背型,异育银鲫,日本白鲫及华南鲤的变种红鲤共１２４个个体的线粒体ＤＮＡ进行了ＲＦＬＰ分析,１４种酶具多态,共计４３种限制性态型,１１种单倍型。     

四个鲫鱼品系线粒体DNA的限制性酶切分析

用差速离心和核酸酶消化法从红鲫 (C auratusredvar .)、湘鲫 [F1hybridsofredcruciancarp (♀ )×commoncarp (♂ ) ]、野鲫 (C auratusauratus)和白鲫 (C auratuscuvieri)的肝组织及白鲫的卵巢中提取和纯化线粒体DNA。用 9种内切酶 (EcoRⅠ、HindⅢ、PstⅠ、BglⅡ、BamHⅠ、XhoⅠ、XbaⅠ、SalⅠ和KpnⅠ )进行单酶酶解 ,经琼脂糖凝胶电泳分析 ,检测出PstⅠ、KpnⅠ和BglⅡ 3种酶在品系间存在限制性片段长度多态性 ,但并未检测出品系内的限制性片段长度多态性。计算出红鲫、湘鲫、白鲫和野鲫的mtDNA大小分别约为 16 19、 16 0 2、 16 6 0和 16 0 6kb。根据限制性酶切片段共享度 ,计算出 4个品系间的遗传距离 ,结果表明存在直接亲缘关系的红鲫与湘鲫之间的遗传差异最小 ,证实了红鲫与子代湘鲫之间mtDNA遵循母系遗传的特性。     

不同赤眼蜂品系ISSR-PCR条件优化及分子鉴定

赤眼蜂Trichogramma不同品系在寄生能力等生物学特性上存在差异,而这些差异可能被用于优良品系的筛选。通过优化影响赤眼蜂不同品系ISSR-PCR的主要参数,建立适于鉴别赤眼蜂不同品系的ISSR-PCR反应体系和扩增程序。在20μL反应体系中,各反应物的最适含量为10×PCR Buffer 2.0μL,MgCl22.0μL（25mmol/L）,dNTPs1.6μL（2.5mmol/Leach）,正反向引物各1μL（25μmol/L）,DNA模板1.0μL,Taq酶0.4μL（2.5U/μL）,和ddH2O。ISSR-PCR的扩增程序为：95℃预变性5min,94℃变性50s,52℃退火1min,72℃延伸1min20s,35个循环,72℃延伸10min。结果表明,在上述优化条件下,采用ISSR-PCR可实现对7个赤眼蜂优良品系的分子鉴定。该研究对于赤眼蜂优良品系的筛选具有潜在的应用价值。     

猕猴桃属植物线粒体DNA片段PCR-RFLP研究初报

猕猴桃属 ( Actinidia)植物 ,全世界有 66个种 ,约 1 1 8个种下分类单位 (变种、变型 ) [1] 。近来通过对猕猴桃属植物的细胞质 DNA研究证实 ,该属植物细胞质 DNA为单亲遗传 ,其线粒体 DNA为严格的母性遗传 [2 ] ,这一单亲遗传特性为研究猕猴桃属植物分类及系统学提供了新的途径。在高等植物中 ,由于线粒体 DNA的高度保守 ,以及重排率高而突变率低 ,加之在植物组织中的含量较低 ,限制了它在系统学研究中的应用 ,不适合属以上高阶元间的比较研究[3 ] ,但线粒体 DNA序列进化速度快 ,在近缘种内或种间显示出了更大的变异性 ,并且线粒体 …     

利用PCR-RFLP技术鉴定传粉榕小蜂隐种混合样品的物种组成

隐种(cryptic species)是指形态上几乎完全相同但遗传组成存在显著分化的物种。在榕树–榕小蜂一对一共生系统中, 传粉榕小蜂隐种的发现对协同进化、物种共存等重要的生态和进化理论提出了严峻的挑战。因为很难从形态上直接区分隐种, 所以, 相关研究中的一个迫切需要解决的问题就是如何快速而准确地鉴定隐种。本文采用PCR-RFLP方法分析了mtDNACOI基因片段, 对木瓜榕(Ficus auriculata)和鸡嗉果榕(F. semicordata)的传粉榕小蜂隐种进行了区分。结果表明为木瓜榕传粉的大果榕小蜂(Ceratosolen emarginatus)存在两个隐种(A和B), 分别包含1个XhoI和1个BssSI酶切位点。将两个隐种的样品按不同比例混合, 提取基因组DNA, PCR扩增mtDNA COI片段, 经XhoI和BssSI分别酶切, 均能通过酶切图谱准确检测出混合样品的隐种组成。鸡嗉果榕小蜂(C. gravelyi)两个隐种的mtDNA COI基因序列也存在较大差别, 分别包含1个BmrI和1个AvaI酶切位点, 隐种混合样品经BmrI和AvaI分别酶切的结果也能准确鉴定混合样品的物种组成。我们的结果表明基于PCR和DNA酶切技术能快速而准确地区分传粉榕小蜂的隐种。     

我国主要地方绵羊品种mtDNA D-loop区PCR-RFLP研究

利用5种限制性内切酶（Hinf I,Msp I,Sau3A I,Xsp I,Taq I）,采用PCR-RFLP技术研究了我国9个地方绵羊品种以及2个引入品种共计83只绵羊个体线粒体DNA D-loop区的多态性。结果表明,我国主要地方绵羊品种线粒体DNA D-loop区存在两种基本单体型,提示我国主要地方绵羊品种起源于两个母系祖先。线粒体DNA D-loop区多态度为0.042 1%,说明我国地方绵羊品种线粒体DNA多态度较为贫乏。
     

PCR对转基因玉米CBH351品系的鉴定检测

成功建立了转基因玉米CBH351（Starlink^TM)的筛选和品系鉴定检测的PCR方法,该方法根据玉米自身IVR基因作为内源特异参照基因来检查模板DNA提取的质量,避免了假阴性结果,设计检测CaMV35S启动子的引物扩增195bp,来对转基因玉米进行筛选检测;并进一步设计转基因玉米CBH351（StarlinkTM)品系转化质粒图谱中CaMV35S启动子和Cry9C边界位置基因特异性引物扩增170bp,以及目标基因Cry9C的右端与CaMV35S终止子的左端边界位置基因特异性引物扩增171bp,以此来鉴定检测CBH351（StarlinkTM)的品系。     

Phylogenetic relationships of silver crucian carp in Carassius auratus complex based on mtDNA analysis

The phylogenetic relationships of Carassius genus subspecies were investigated based on the data of the variability of nucleotide sequences of the mtDNA cytochrome b (cyt b) and control region (CR). Dendrograms constructed based on the BA, ML, NJ, and MP methods revealed five clusters of the congruent topologies that substantially corresponded to geographical localities and taxonomic conception of the C. auratus complex. An analysis of two mtDNA fragment topologies demonstrated that the island forms of Japanese crucian carps C. cuvieri and C. auratus langsdorfii diverged later compared to the divergence of continental C. auratus forms (4.0?C4.5 mln years ago, by molecular calibration). Among the continental silver crucian carps, C. a. gibelio forms two clusters corresponding to two phylogroups with a mean uncorrected genetic distance p = 0.044. The genealogical combination of haplotypes with the first C. a. gibelio phylogroup was observed in C. auratus clade. According to the data of mtDNA analysis, these subspecies represent sister lineages with a level of intergroup divergence of p = 0.022?C0.036. No genetic differences were observed between diploid (except for the two C. a. gibelio phylogroups) and polyploid C a. auratus, as well as monophyly in polyploid forms. New approaches based on a comparative study of the nuclear markers might help to unravel the origin of gynogenetic forms and phylogenetic relationships within the C. auratus complex.     

Molecular phylogenetic systematics of twelve species of Acipenseriformes based on mtDNA ND4L -ND4 gene sequence analysis

Acipenseriformes is an endangered primitive fish group, which occupies a special place in the history of ideas concerning fish evolution, even in vertebrate evolution. However, the classification and evolution of the fishes have been debated. The mitochondrial DMA (mtDNA) ND4L and partial A7D4 genes were first sequenced in twelve species of the order Acipenseriformes, including endemic Chinese species. The following points were drawn from DNA sequences analysis: (i) the two species of Huso can be ascribed to Acipenser; (ii) A. dabryanus is the mostly closely related to A. sinensis, and most likely the landlocked form of A. sinensis; (iii) genus Acipenser in trans-Pacific region might have a common origin; (iv) mtDNA ND4L and ND4 genes are the ideal genetic markers for phylogenetic analysis of the order Acipenseriformes.     

Carbohydrate metabolism of goldfish (Carassius auratus L.)

Summary The effect of hypoxia was studied in cold (15°C) and warm (30°C) acclimated goldfish. The hypoxic thresholds, defined as the lowest sustainablePO₂ were found to be 1.6 and 4.0 kPa O₂ at, respectively, 15°C and 30°C. At these levels the fish did not loose either weight or appetite over a 2-months period. While during starvation under normonic conditions a significant weight loss and breakdown of lactate dehydrogenase (90%) was observed, no such changes were found in fed hypoxic animals. In red lateral muscle, white epaxial muscle and liver of goldfish from 4 differently acclimated groups the maximal activities were measured of: glycogen phosphorylase, hexokinase, malate dehydrogenase, glycerol-3-P dehydrogenase, glucose-6-P dehydrogenase, malic enzyme, succinate oxidase, pyruvate carboxylase, phosphoenol-pyruvate carboxykinase, fructose-bisphosphatase and glucose-6-phosphatase. Thermal compensation, according to Precht's typology, was predominantly observed in red muscle and to a lesser extent in white muscle. The liver glucose-6-P dehydrogenase showed a strong inverse response, which points to enhanced synthetic activity at the higher temperature. Hypoxia acclimation exerted weaker responses at 15°C than at 30°C. Changes in liver enzyme activities suggest depressed protein synthesis and enhanced gluconeogenesis in hypoxic animals. In muscle of 30°C-acclimated goldfish hypoxia induces a significant increase of succinate oxidase activity, indicating adaptation of the aerobic energy metabolism. The occurrence of pyruvate carboxylase, never before observed in vertebrate muscle, probably plays an important role in pyruvate catabolism. Because its action produces oxalo-acetate, the enzyme may stimulate pyruvate oxidation and thus prevent early lactate accumulation. Since all gluconeogenic enzymes were shown to be active in goldfish muscle, the possible occurrence of gluconeogenesis in muscle (albeit at low rate) must be accepted. Enzyme activities in goldfish muscle were compared with literature data for a number of other fish species. This comparison indicates that maximal glycolytic flux in goldfish muscle tissue is rather low, although muscular glycogen levels are very high. It is suggested that this is part of the gold-fish's strategy to cope with hypoxia.     

Molecular phylogenetic systematics of twelve species of Acipenseriformes based on mtDNA ND4L -ND4 gene sequence analysis

Acipenseriformes is an endangered primitive fish group, which occupies a special place in the history of ideas concerning fish evolution, even in vertebrate evolution. However, the classification and evolution of the fishes have been debated. The mitochondrial DNA (mtDNA) ND4L and partial ND4 genes were first sequenced in twelve species of the order Acipenseriformes, including endemic Chinese species. The following points were drawn from DNA sequences analysis: (i) the two species of Huso can be ascribed to Acipenser; (ii) A. dabryanus is the mostly closely related to A. sinensis, and most likely the landlocked form of A. sinensis; (iii) genus Acipenser in trans-Pacific region might have a common origin; (iv) mtDNA ND4L and ND4 genes are the ideal genetic markers for phylogenetic analysis of the order Acipenseriformes.     

Hepatic accumulation and effects of microcystin-LR on juvenile goldfish Carassius auratus L

After intraperitoneal injection of microcystin-LR (MC-LR) (125 microg kg(-1) body wt.), the concentration of MC-LR in the liver of juvenile goldfish Carassius auratus (30 g body wt.) was assayed by a modified protein phosphatase inhibition method. A temporary accumulation occurred from 3 to 48 h post-injection, followed by a significant decrease between 48 and 96 h. Under our experimental conditions, contamination by MC-LR did not change ionic homeostasis, as attested by blood osmolality values and gill Na(+)/K(+) ATPase activity. Light microscopy observations revealed lesions and cellular necrosis progression, which was concomitant with an increase in enzyme activity of plasma aspartate aminotransferase (AspAT), alanine aminotransferase (AlaAT) and L-lactate dehydrogenase (LDH) and with a decrease of hepatic glutathione-S-transferase (GST) activity. Structural alterations and enzymatic activity modifications became significant within 24 h post-injection. Recovery of hepatocytes on day 21 after MC-LR injection was evident, together with a decrease in the MC-LR equivalent content of the liver.     

Morpho-functional characterization of the goldfish (Carassius auratus L.) heart

Using morphological and physiological approaches we provided, for the first time, a structural and functional characterization of Carassius auratus L. heart. Besides to the classical four chambers, i.e. sinus venosus, atrium, ventricle, bulbus, we described two distinct structures corresponding to the atrio-ventricular (AV) region and the conus arteriosus. The atrium is very large and highly trabeculated; the ventricle shows an outer compacta, vascularized by coronary vessels, and an inner spongiosa; the bulbus wall is characterized by a high elastin/collagen ratio, which makes it extremely compliant. Immunolocalization revealed a strong expression of activated eNOS-like isoforms both at coronary endothelium and, to a lesser extent, in the myocardiocytes and the endocardial endothelium (EE). The structural design of the heart appears to comply with its mechanical function. Using an in vitro working heart preparation, cardiac performance was evaluated at different filling and afterload pressures. The hearts were very sensitive to filling pressure increases. Maximum Stroke volume (SV=1.08 ± 0.09 mL/kg body mass) was obtained with an input pressure of 0.4 kPa. The heart was not able to sustain afterload increases, values higher than 1.5 kPa impairing its performance. These morpho-functional features are consistent with a volume pump mechanical performance.