鱼类线粒体DNA研究新进展

线粒体DNA是分子生物学研究中的一个热门领域,已成为鱼类进化生物学和群体遗传学研究的重要分子遗传标记。本文对鱼类线粒体DNA分子生物学的最新研究进展进行了较详细的阐述。重点介绍鱼类线粒体DNA全序列的研究进展、组成及特征,鱼类线粒体DNA非编码区结构研究进展,鱼类线粒体DNA多态性及其主要的检测方法;综述了最近有关鱼类线粒体DNA在鱼类系统学、种间杂交渐渗、种群识别、起源和进化、地理分化等研究中的应用情况。     

鱼类线粒体DNA及其研究进展

鱼类是脊椎动物中最原始而在种属数量上又最占优势的类群,其起源复杂,分布广泛,拥有丰富的遗传多样性。鱼类线粒体DNA(mitochondrial DNA,mtDNA)同其他脊椎动物的mtRNA一样,呈共价闭合环状,是细胞核外具自主复制、转录和翻译能力的遗传因子。与核DNA相比,鱼类mtDNA具有分子较小、结构简单、进化速度快、遗传相对独立性和母系遗传等特点,是一个相对独立的复制单位。由于鱼类线粒体DNA具有上述特点,以mtDNA作为分子标记,探讨鱼类的群体遗传结构与系统演化,已成为鱼类分子群体遗传学和系统学研究中的热点。综述了鱼类mtDNA的结构特征、进化和多态性检测方法及其在鱼类分子群体遗传学和鱼类系统学研究中的应用。     

长江流域■种群遗传多样性和遗传结构分析

鱼类群体遗传学研究主要集中在经济鱼类或濒危物种,然而一些经济价值较低的物种的遗传结构却甚少关注。因此,研究选择了经济价值较低的■(Hemiculter leucisculus),共计323尾个体分别来自13个长江流域及其附属湖泊的自然群体。通过扩增线粒体DNA Cytb基因序列片段(1100 bp),以探讨■种群遗传结构和遗传多样性。遗传多样性分析呈现出高单倍型多样性和高核苷酸多样性的模式,表明该种群在长江流域较为稳定。另外,基于线粒体细胞色素b基因的系统发育分析,显示■有5个线粒体谱系(谱系A-F)组成。中性检验和核苷酸错配分布分析均显示谱系A、B、E、F曾经历过种群扩张,并且呈现从上游向中游扩张的规律。谱系间较高且显著的遗传分化指数和显著的系统进化关系,均表明谱系A-F之间存在明显的遗传分化,暗示长江流域可能至少存在4个不同线粒体DNA水平上的种。■种群的遗传结构和多样性可能受到了长江流域特定格局的影响。     

分子进化与中立说

生物进化的研究向来是以生物的表型为对象的,如蕨类植物的维管束,马的脚趾,哺乳动物的齿冠等,都是用古生物学方法来研究的。生物的进化当然是由自然选择理论来说明的,不过这些都是定性的说明而已。 用定量方法研究进化论,是从群体遗传学的理论研究开始的。种的进化不仅是个体水平的变化,主要是整个群体的遗传变化。例如某个种的一个基因变化     

草鱼野生和养殖群体间遗传变异的微卫星分析

运用微卫星标记对江苏境内草鱼(Ctenopharyngodonidella)一个野生群体(邗江群体)和两个养殖群体(淡水中心群体和无锡前洲群体)遗传多样性进行了分析。在10个座位中,每个座位检测到的等位基因数2~8个。有效等位基因数、多态信息含量、期望杂合度、平均表观杂合度均以邗江草鱼野生群体最高,分别为3·9、0·5068、0·6939、0·7;无锡前洲草鱼养殖群体最低,分别为2·2、0·1796、0·5235、0·5286;淡水中心草鱼养殖群体各参数均介于两者之间,分别为3·5、0·2902、0·5418、0·5429。以上结果表明:草鱼野生群体遗传多样性更为丰富,而草鱼养殖群体存在杂合度降低,遗传多样性下降的现象。邗江草鱼野生群体与淡水中心草鱼养殖群体和无锡前洲草鱼养殖群体间遗传分化系数分别为0·219和0·246,而两个草鱼养殖群体间遗传分化系数为0·034。这表明草鱼野生群体与草鱼养殖群体间分化严重,而草鱼养殖群体间分化微弱。各座位分化程度的χ2检验结果表明,10个座位中有GM18、MFW1-1、MFW1-2三个座位群体间分化达到极显著水平,GM03-2、MFW5两个座位群体间分化差异显著,其他座位分化不显著。针对每个座位对各群体进行Hardy-Weinberg平衡检验发现:由于草鱼养殖群体在GM03-1、GM03-2、GM18三个位点杂合子缺失,草鱼野生群体在位点GM19杂合子过剩而严重偏离平衡。实验表明:近交容易引起草鱼遗传多样性下降,纯合速度加快。     

两个不同的人工雌核发育草鱼群体基因组DNA的RAPD分析

采用随机扩增多态性DNA(RAPD)技术对连续两代人工诱导雌核发育草鱼群体和一代人工诱导雌核发育草鱼群体的群体内的遗传相似度、遗传距离以及群体多样性进行了分析 ,并用一个随机取样的普通草鱼群体作比较。用 2 6个多态性引物在 3个群体中共检测到了 2 91个扩增位点 ;在 3个群体中检测到的位点数分别为 2 6 5、2 72和2 82。其中多态性位点数分别为 15、19和 81。遗传学统计分析结果表明 :3个群体的多态位点比例分别为 5 6 6 %、6 99%、2 8 72 % ;香农表型多样性指数分别为 0 170 2、0 316 9和 0 84 5 0 ;按照Nei指数统计的三个群体的遗传相似度分别为 0 985 1、0 982 0、0 9114。这些结果表明 :两个雌核发育草鱼群体的遗传多样性远低于普通草鱼群体 ,而其群体的基因组DNA同质性远高于普通草鱼。而在两个雌核发育草鱼群体中 ,两代雌核发育群体的遗传多样性要低于一代雌核发育群体的遗传多样性 ;而群体的遗传纯合度则前者高于后者。     

鱼类线粒体DNA及其在分子群体遗传研究中的应用

鱼类是脊椎动物亚门中种属数量最多的类群,分布广泛,起源复杂,拥有丰富的遗传多样性.多种自然和人为因素对鱼类遗传资源存在不同程度的作用,对鱼类生存和进化有重要影响.采用分子手段探讨鱼类遗传资源现状,可为遗传育种、鱼类进化研究和遗传资源保护等提供一定科学依据.以鱼类线粒体DNA(mtDNA)为代表的分子标记技术已被用于研究鱼类群体遗传结构及其与影响因素间的关系.本文综述了鱼类mtDNA的结构特征及其在鱼类分子群体遗传研究中的应用,对了解和运用mtDNA等分子标记研究鱼类群体遗传具有一定参考价值.     

罗氏沼虾不同群体线粒体COI基因的序列差异和遗传标记研究

利用PCR方法扩增获得罗氏沼虾（Macrobrachiumrosenbergii）线粒体DNA的COI基因,测定该基因片段序列。分析了罗氏沼虾缅甸原种F1代、江苏养殖群体和广西选育F2代3个群体共17只个体的序列核苷酸位点差异和遗传多态。结果表明,缅甸原种F1代遗传多样性最为丰富,江苏养殖群体和广西选育群体的遗传多样性相对贫乏。在长度为498bp的基因片段中,共检测到10个多态性核苷酸位点（占2.01%）,17只个体具有5种基因型,3群体各自的平均核苷酸位点差异分别为0.88%、0.07%和0。UPGMA分子系统聚类树显示,江苏养殖群体和广西选育群体的遗传关系最近,其单倍型混杂聚成一支,而缅甸原种F1群体相对独立为另外一支。COI基因可以作为区分两分支群体的遗传标记。     

罗氏沼虾3个群体线粒体COⅠ基因的序列差异和遗传标记研究

利用PCR方法扩增获得罗氏沼虾(Macrobrachium rosenbergii)线粒体DNA的COⅠ基因,测定该基因片段序列.分析了罗氏沼虾缅甸原种F1代、江苏养殖群体和广西选育F2代3个群体共17只个体的序列核苷酸位点差异和遗传多态.结果表明,缅甸原种F1代遗传多样性最为丰富,江苏养殖群体和广西选育群体的遗传多样性相对贫乏.在长度为498 bp的基因片段中,共检测到10个多态性核苷酸位点(占2.01%),17只个体具有5种基因型,3群体各自的平均核苷酸位点差异分别为0.88%、0.07%和0.UPGMA分子系统聚类树显示,江苏养殖群体和广西选育群体的遗传关系最近,其单倍型混杂聚成一支,而缅甸原种F1群体相对独立为另外一支.COⅠ基因可以作为区分两分支群体的遗传标记.     

转基因动物的基础与应用研究

转基因动物研究的基础：１．经典遗传学：揭示了遗传基因的染色体几何位点与生物表型性状的形式对应关系。生物个体体现为物种内全套基因不同等位基因的组合。２．分子遗传学：阐明了基因到性状是核酸到蛋白的信息流调控过程,即操作结构模型和中心法则的研究范畴。３．结构遗传学：基因组结构（遗传语法）、功能（发育调控）与演变（物种进化）过程构成物种进化与发育自组织的结构遗传学基础。进化是基因组的信息化增长结构化分层、     

Genetic tags applied to the European hake, Merluccius merluccius (L.)

Three biochemical gene markers test the hypothesis that the European hake, Merluccius merluccius (L.), along the west European continental shelf are one race. The three polymorphic loci were serum transferrin (Tf), eye vitreous fluid butyric esterase (Es) and liver superoxide dismutase (Sod). Five transferrin alleles, three esterase alleles and two superoxide dismutase alleles were identified. Heterogeneity tests on genotype frequency distribution for twelve areas ranging from Norway to Biscay revealed no significant variation. The results using these genetic tags are consistent with the unit race hypothesis for hake throughout the sea areas sampled.     

Genetic tags applied to the European hake, Merluccius merluccius (L.)

Three biochemical gene markers test the hypothesis that the European hake, Merluccius merluccius (L.), along the west European continental shelf are one race. The three polymorphic loci were serum transferrin (Tf), eye vitreous fluid butyric esterase (Es) and liver superoxide dismutase (Sod). Five transferrin alleles, three esterase alleles and two superoxide dismutase alleles were identified. Heterogeneity tests on genotype frequency distribution for twelve areas ranging from Norway to Biscay revealed no significant variation. The results using these genetic tags are consistent with the unit race hypothesis for hake throughout the sea areas sampled.     

草鱼连续两代的雌核发育群体及湘江流域群体基因组DNA的微卫星比较分析

对湘江流域草鱼群体,一代及连续两代极体型人工诱导雌核发育草鱼群体基因组DNA的微卫星引物统计分析结果表明:湘江流域草鱼群体存在一定程度的遗传多态性;大多数被检测的微卫星位点上存在两个以上的等位基因,但遗传多样性程度较低。一代人工诱导雌核发育草鱼群体中个体的基因位点已基本纯合,但就整个群体而言,个体之间的基因型还不完全一致,表现出一定的多态性。连续两代人工诱导雌核发育草鱼群体中不仅所检测个体的基因位点已完全纯合,并且各个体的基因型也完全相同。这些观察结果说明所检测的两代人工诱导雌核发育草鱼群体是纯合的,经连续两代人工诱导雌核发育可能建立起草鱼纯系。该实验结果还发现不仅草鱼的微卫星位点上存在等位基因的多态性,而且微卫星位点本身也存在多态性;在人工诱导草鱼雌核发育的过程中不仅存在微卫星等位基因快速丢失的现象,而且也存在微卫星位点丢失的现象。因此,加强对自然水体中草鱼种质资源多样性的保护和利用各种现代生物学技术纯化、筛选和组合优良性状基因,是草鱼遗传育种中同样重要和不可或缺的两个方面。     

Comparative antioxidant enzyme study in freshwater fishes. I. Distribution of superoxide dismutase, peroxide-decomposing enzymes and lipid peroxidation in herbivorous fishes

The activities of Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase were comparatively studied in organ homogenates from two herbivorous fishes, viz., the grass carp and the silver carp. 2. The protein contents and lipid peroxidation of organ homogenates were also compared. The comparative measurements primarily provide control values for subsequent toxicological examinations. 3. The highest total superoxide dismutase activities were found in the kidney, spleen and liver in the grass carp, and in the kidney and liver in the silver carp. 4. The antioxidant enzyme activities and other parameters of the organ homogenates appear to be independent of the feeding mode, but are rather characteristic of the fish variety.     

草鱼雌核发育后代不同群体的微卫星遗传分析及指纹识别

以紫外线灭活的团头鲂精子激活草鱼卵子,冷休克抑制第二极体排出的方法诱导出长江水系优良F2代草鱼减数雌核发育子代。在后代中不仅存在雌核发育后代,还存在草鲂杂交后代,雌核发育后代的体型与草鱼一致,而草鲂杂交后代的体型介于草鱼与团头鲂之间。Partec CyFlow倍性分析仪测定结果显示:普通草鱼与雌核发育草鱼的相对DNA含量分别为23.01和22.72,二者的DNA含量接近;而高体型子代的相对DNA含量为25.38,介于草鱼与团头鲂（DNA含量28.21）之间,属于草鲂杂交后代。选取17个微卫星标记对草鱼群体、雌核发育草鱼群体和草鲂杂交后代的遗传多样性进行了检测,共检测出59个等位基因,其中43.18个有效等位基因。草鱼对照群体、草鲂杂交后代和雌核发育草鱼群体的平均等位基因依次为3.57、2.86和2.79,平均有效等位基因依次为2.93、2.37和1.96,平均期望杂合度在依次为0.6502、0.5573和0.3775,多态信息含量（PIC）平均值依次为0.5738、0.4649和0.3791。与草鱼对照群体相比,雌核发育草鱼群体的遗传多样性显著下降,表明通过减数雌核发育方法可获得纯合性较高的草鱼个体。构建了草鱼后代不同群体的DNA指纹模式图,筛选到不同群体的9个特异微卫星标记,为草鱼优良群体的选育提供了基础资料。     

应用微卫星多态分析四个鲤鱼群体的遗传多样性

选择12个斑马鱼功能基因的微卫星标记和12个鲤鱼微卫星标记,检测黑龙江鲤(Cyprinus carpiohaematopterusTemminck et Schlegel)、散鳞镜鲤(C.carpio)、荷包红鲤抗寒品系(C.carpiovar.wuyuanensis)、松浦鲤(C.carpioSongpu carp)的群体遗传多样性。共检测到3882个扩增片段,长度为126～489bp,在群体间扩增出1～5个等位基因不等,共计59个等位基因,平均等位基因2.46个。数据经PHYLIP V3.6软件估算和MEGA3软件作图,确立4个群体间的亲缘关系。并应用Bootstrap检验估计系统树中节点的自引导值,并进行了系统发生分析。结果表明①4个群体检测的有效等位基因数都在55个以上,平均观测杂合度为0.36～0.43,平均期望杂合度为0.49～0.53,平均多态信息含量为0.21～0.25,说明这几个群体多态性属于中度偏高水平,遗传多样性较高;②群体间相似系数都在0.84以上,遗传距离较近,为0.067～0.170,与前人研究结果一致。聚类分析显示,松浦鲤与散鳞镜鲤亲缘关系最近,荷包红鲤抗寒品系与它们的亲缘关系较黑龙江鲤更近;③在3个斑马鱼功能基因相关的微卫星位点上,黑龙江鲤缺失特异扩增条带,这可能与几种鱼类的育成史有关。     

Isozyme expression in F1 hybrids between carp and goldfish

Interspecific genetic differences in malate dehydrogenase (MDH), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and esterase (EST) isozymes in carp (Cyprinus carpio) and goldfish (Carassius auratus) were used to examine the allelic expressions in the hybrid between these species. A unique liver SOD and muscle LDH phenotype unambiguously identifies all presumed hybrid individuals. There was no evidence of F₂ or backcross phenotypes in hybrid individuals. Liver MDH and EST phenotypes in hybrids show a preferential expression of goldfish isozymes. Variation in the levels of carp liver MDH isozymes may result from the polymorphism of a regulatory mutation affecting isozyme expression, leading to gene silencing after duplication.This work was supported through NSERC (Canada) grants to James P. Bogart and John F. Leatherland.     

Comparative antioxidant enzyme study in freshwater fish with different types of feeding behaviour

Comparative studies were made of the activities of Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase in organ homogenates from two herbivorous (grass carp and silver carp) and three omnivorous fish (barbel, crucian carp and common carp). The protein contents and lipid peroxidation of the organ homogenates were also compared. These comparative measurements provided control values for subsequent toxicological examinations. For the herbivorous fish, the highest total superoxide dismutase activity was observed in the kidney, followed in turn by the liver or spleen. In contrast, the highest total superoxide dismutase activity in the barbel was found in the roe, in the crucian carp in the liver, and in the common carp in the liver too. The quantitative distributions of the two peroxide metabolism enzymes glutathione peroxidase and catalase in the organ homogenates present very varied pictures. The results of the lipid peroxidation are similarly difficult to fit into one framework, even for fish following the same diet. It appears that the enzyme activities mentioned in points 4 and 5, together with the lipid peroxidation, depend not on the species, but on the variety.     

Induced Gynogenesis in Grass Carp (Ctenopharyngodon idellus) Using Irradiated Sperm of Allotetraploid Hybrids

Grass carp (Ctenopharyngodon idellus) eggs were activated by UV-irradiated diploid sperm of allotetraploid hybrids derived from red crucian carp (♀)?×?common carp (♂) and then duplicated by cold shock in 4-6°C water for 10-12 min. Different cold shock initiation times resulted in two types of diploid gynogenetic grass carp: meiotic gynogenetic (meiG) and mitotic gynogenetic (mitG). Over a 5-year period, a total of 17,170 meiG and 1,080 mitG fry were produced and 6,862 meiG and 372 mitG grass carp survived. The gynogenetic fish were confirmed by morphological characteristics, chromosome examination, and microsatellite DNA analysis. The morphological traits of the gynogenetic grass carp were similar to those of wild diploid grass carp. Normal gynogenetic fish were identified as diploid with 48 chromosomes by chromosomal metaphases examination, while nonviable abnormal embryos were detected as haploid with 24 chromosomes. Microsatellite DNA analysis indicated that after one generation of gynogenesis, the genetic purity of meiG and mitG grass carp was significantly increased over that of wild grass carp. In addition, both meiG and mitG grass carp groups were 100% female, and 88% of these showed normal ovary development. Thus, the sex determination mechanism in female grass carp was homogamety. The ability to establish pure all-female groups of meiG and mitG grass carp should be a valuable contribution to both fish genetics and grass carp breeding.     

The activity of pro- and antioxidant systems in the liver of freshwater fishes during different seasons

The content of lipid peroxidation products--diene conjugates, lipid hydroperoxides, thiobarbituric acid reactive substances (TBARS), vitamins A, E and carotenoids and the activity of antioxidant enzymes--superoxide dismutase, glutathione peroxidase and catalase in the liver of freshwater fishes of different species (silver carp, grass carp and common carp) in different seasons have been studied. It was established the activity of antioxidant defence system in the liver of fish depends significantly on the season and fish species. In particular, the content of lipid peroxidation products in the liver of freshwater fishes at the beginning of winter and spring was significantly higher compared to their content at the beginning of summer and autumn. The superoxide dismutase and glutathione peroxidase activities in the liver of these fish species at the beginning of winter and spring were significantly lower than at the beginning of summer and autumn while the seasonal changes of catalase activity in the liver of fish are expressed insignificantly. The content of vitamins E, A1, A2 and carotenoids in the liver of fishes of different species at the beginning of winter and spring was significantly lower than at the beginning of summer and autumn. The content of lipid peroxidation products and vitamins E, A1 and A2 in the liver of common carp is significantly lower than in the liver of silver carp and grass carp and species differences in antioxidant enzymes activity are insignificant.