鸭类mtDNA限制性酶切图谱的比较研究

本文报道了番鸭、建昌鸭、杂交鸭(♀建昌鸭×(?)番鸭)和北京鸭的mtDNA限制性酶切图谱。限制性内切酶HindⅢ、PsiⅠ、BamHⅠ和EcoRⅠ在番鸭mtDNA上分别有(?)、2、2和1个酶切位点。建昌鸭、杂交鸭和北京鸭的mtDNA限制性酶切图谱完全一致。以上4种内切酶在这3种鸭子mtDNA上分别有5、4、2和1个位点。比较研究4种限制性图谱,我们得到三点结论:(1)鸭类mtDN种间差异程度很高;(2)家鸭很可能不存在mtDNA种内异质现象;(3)鸭类mtDNA为母性遗传。     

三种鱼mtDNA的限制性内切酶分析

鱼类线粒体DNA(mtDNA)的限制性酶切图谱分析,对于探讨鱼类的起源和演化等方面均有十分重要的意义。但是目前有关鱼类mtDNA酶切图谱的研究较少,这主要是受方法学的限制。为此我们改良了一种鱼类mtDNA的提取法,对鲤科的草鱼(Ctenopharyngodon idellus)、鲢鱼(Hypophthalmichthys molitrix)和鳙鱼(Aristi-chthys nobilis)的mtDNA进行了限制性内切酶酶切分析。     

鳗鲡线粒体DNA的研究

本文采用差速离心法及RNase消化法制备并纯化了级鲡(Anguilla Japonica Temmink et Schlegel)肝脏线粒体DNA(mtDNA),用8种限制性内切酶对mtDNA进行了分析．BgП、Xhoh、Psth、EcoRL、BamHI、HindШ、Bgli、Xbal在鳗鲡mtDNA分子上分别具0, 1, 3, 5, 2, 7, 2, 4个切点．mtDNA分子量约10.16x10⁶道尔顿,大小为16.44kb.根据鳗鲡肝mtDNA的单酶及双酶完全酶解片段的大小,建立了鳗鲡mtDNA的限制性酶切图谱．     

达乌尔鼠兔和高原鼠兔肝脏线粒体DNA的限制性内切酶图谱的比较研究

达岛尔鼠兔肝细胞线粒体DNA(mtDNA),经限制性内切酶HindI、HindII,EcoRI和BamHI酶切后分别产生5、4、3、2个片段。通过琼脂糖凝肢电泳对这些片段进行测定,并画出其酶切图谱。高原鼠兔肝细胞mtDNA经限制性内切酶EcoR I和BamH 1酶切后,分别产生4、2个片段,对其片段的分子量也进行了测定。测定结果,达乌尔鼠兔肝细胞mtDNA的分子量为10.25MD(兆道尔顿)．大小为16.25kbp(千碱基对);高原鼠兔肝细胞mtDNA的分子量为9.31MD,大小为l5.066kbp。并对两种鼠免的限制性内切酶片段进行了比较讨论。     

鸡肝脏线粒体DNA的限制图谱

本文用六种限制性内切酶对鸡肝脏线粒体DNA(mtDNA)进行了酶解。Eco RⅠ、Bam HⅠ、SalⅠ、HindⅢ、BglⅠ在鸡肝mtDNA上分别有2、2、3、4、4个切点,BglⅡ不能切割鸡肝mtDNA。根据鸡肝mtDNA的单酶、双酶完全酶解以及部分酶解片段的分子量,建立了鸡肝mtDNA的限制图谱。     

鳜及大眼鳜线粒体DNA比较研究

采用10种限制性内切酶HindⅢ、EcoRⅠ、BglⅠ、BglⅡ、BamHⅠ、PvuⅡ、PstⅠ、SalⅠ、XbaⅠ、XhoⅠ对鳜及大眼鳜肝脏线粒体DNA（mtDNA）进行了分析。鳜鱼mtDNA分子量为9.703×106u,大小为16．19kb;大眼鳜mtDNA分子量为9．603×106u,大小为16．02kb。根据单、双酶切结果构建了鳜及大眼鳜mtDNA10种酶限制性酶切图谱,并对两种鱼的mtDNA酶切图谱进行了比较。     

长江中游水系鲢和草鱼群体mtDNA遗传变异的研究

采用PCR技术进行了长江中游鲢和草鱼四个地理群体的线粒体DNA（mtDNA)限制性片段长度多态性（RFLP）的研究。四个地理群体包括长江中游的湖北嘉鱼和江西瑞昌两个地理群体,长江中游的两大支流江汉和湘江群体。PCR技术扩增出mtDNA ND5-ND6基因,选用10种限制性内切酶对PCR产物进行酶切。从鲢中共检出18种单倍型,在草鱼中没有发现多态现象,只有一种单倍型存在。进一步地证实了长江鲢的遗传多样性比草鱼的要丰富得多,与这两种鱼类在长江现有生物量成反比的反常现象。     

雌核发育草鱼同工酶分析

运用聚丙烯酰胺凝胶电泳技术,分析了雌核发育草鱼尾鳍中LDH、EST、SOD三种同工酶的酶谱。研究结果表明雌核发育草鱼各个体间及与普通草鱼与LDH、EST同工酶谱上表达是一致的。雌核发育草鱼中SOD酶谱与普通草鱼存在有明显差异。在雌核发育草鱼SOD酶谱区段1中检测到一条特殊的酶带(暂称之为SOD-G),SOD-G在所检测的6尾雌核发育草鱼中普遍存在,但在普通草中未检测到,初步认为SOD-G可以作为雌核发育草鱼群体的生化遗传标记。     

FMMU白化豚鼠线粒体DNA RFLP分析研究

目的研究FMMU白化豚鼠的mtDNA,并与花色豚鼠mtDNA进行多态性分析比较,以确定其独特的生物学特性是否与mtDNA相关。方法用碱变性法提取FMMU白化豚鼠以及花色豚鼠的mtDNA,并用AvaⅠ、BalⅠ等12种限制性内切酶进行酶切和限制性片段长度多态性分析。结果与结论FMMU白化豚鼠mtDNA和花色豚鼠mtDNA的相对分子质量相同,约为16.7×103;FMMU白化豚鼠与花色豚鼠两品系的mtDNA经AvaⅠ、BalⅠ等内切酶酶切后有3-8个酶切位点,酶切图谱完全相同,经RFLP分析FMMU白化豚鼠与花色豚鼠的mtDNA之间缺乏多态性。本实验没有发现FMMU白化豚鼠的独特的生物学特性与mtDNA相关。     

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。     

草鱼和鲤鱼线粒体 DNA 的分离纯化及其 COI 基因的分子克隆

本文报道配合使用差速离心和DNaseI核酸酶处理等步骤,从草鱼和鲤鱼新鲜肝组织分离线粒体DNA(mtDNA)的实验方法。这种方法经济简便,纯化的mtDNA产率多,纯度高,经限制性内切酶消化后进行琼脂糖凝胶电泳分离,可以得到清晰的DNA片段谱带,并可直接用于构建酶切图谱和线粒体基因的分子克隆。用这样的mtDNA,我们已克隆了草鱼和鲤鱼的细胞色素氧化酶亚基I基因(COI基因)。     

几种鱼肝线粒体基因组的研究

我们用简易方法提纯了武昌鱼、乌鱼、草鱼和白鲢肝脏线粒体(mt)DNA。武昌鱼mt DNA经五种限制酶BamHI、BglⅡ、BglI、EcoRI和HindⅢ单酶完全酶解分别产生2、2、3、3和3个片段。乌鱼肝mt DNA用EcoRI、BamHI、pstI和BglⅡ单酶解分别得到1、2、2和2个片段。草鱼mt DNA亦作相应的酶解。所有各片段经琼脂糖凝胶电泳测定出它们的分子量(MW):武昌鱼肝mt DNA的MW10.2×10~6道尔顿,长约16.6kb;乌鱼肝mt DNA MW9.99×10~6道尔顿,长16.2kb;草鱼mt DNA的分子量10.13×10~6,长16.22×10kb。用七对限制酶双酶全酶解构建出武昌鱼mt DNA五种限制酶图谱。用六对限制酶双酶解构建出乌鱼mt DNA四种限制酶图。以酵母线粒体15S rRNA为探针对武昌鱼、白鲢鱼和乌鱼mt DNA12S rRNA基因进行了初步定位。     

Collation of data on the ploidy levels and mitochondrial DNA phylogenetic lineages in the silver crucian carp Carassius auratus gibelio from Far Eastern and Central Asian populations

The distribution of the diploid and triploid forms and the correspondence between ploidy and mitochondrial DNA (mtDNA) phylogenetic lineages of the silver crucian carp have been studied in Far Eastern water bodies and the Syr Darya River. Both diploid and triploid forms have been found in large river systems (the Amur, Suifun, Tumangan, and Syr Darya river basins). Only the diploid form has been detected in lakes of Bol'shoi Pelis Island (Peter the Great Bay of the Sea of Japan), Sakhalin Island, and the Kamchatka River basin (the Kamchatka Peninsula). It has been confirmed that there are two mtDNA phylogroups in the silver crucian carp in the area studied. Both mtDNA phylogenetic lineages are present in the Suifun and Tumangan river basins. Only one mtDNA phylogroup (characteristic of the gynogenetic form) has been detected in two samples from the Amur River and in the Syr Darya population. The other mtDNA phylogroup is predominant in insular populations and in Kamchatka. The gynogenetic form carries only mtDNA phylogroup I, whereas both phylogroups have been found in diploid bisexual fish. The existence of only two mtDNA phylogroups substantially differing from each other indicates that the gynogenetic form has emerged from the diploid form only once and evolved independently for a long time after that. The absence of haplotypes transitional between the two mtDNA phylogroups suggests that the secondary contact between the gynogenetic and bisexual forms in continental populations occurred within recent historical time. The obtained data confirm that genetic (though asymmetric) exchange between the two forms is possible, which explains the high morphological and, probably, genetic similarity between them.     

Collation of data on the ploidy levels and mitochondrial DNA phylogenetic lineages in the silver crucian carp <Emphasis Type="Italic">Carassius auratus gibelio</Emphasis> from Far Eastern and Central Asian populations

The distribution of the diploid and triploid forms and the correspondence between ploidy and mitochondrial DNA (mtDNA) phylogenetic lineages of the silver crucian carp have been studied in Far Eastern water bodies and the Syr Darya River. Both diploid and triploid forms have been found in large river systems (the Amur, Suifun, Tumangan, and Syr Darya river basins). Only the diploid form has been detected in lakes of Bol’shoi Pelis Island (Peter the Great Bay of the Sea of Japan), Sakhalin Island, and the Kamchatka River basin (the Kamchatka Peninsula). It has been confirmed that there are two mtDNA phylogroups in the silver crucian carp in the area studied. Both mtDNA phylogenetic lineages are present in the Suifun and Tumangan river basins. Only one mtDNA phylogroup (characteristic of the gynogenetic form) has been detected in two samples from the Amur River and in the Syr Darya population. The other mtDNA phylogroup is predominant in insular populations and in Kamchatka. The gynogenetic form carries only mtDNA phylogroup I, whereas both phylogroups have been found in diploid bisexual fish. The existence of only two mtDNA phylogroups substantially differing from each other indicates that the gynogenetic form has emerged from the diploid form only once and evolved independently for a long time after that. The absence of haplotypes transitional between the two mtDNA phylogroups suggests that the secondary contact between the gynogenetic and bisexual forms in continental populations occurred within recent historical time. The obtained data confirm that genetic (though asymmetric) exchange between the two forms is possible, which explains the high morphological and, probably, genetic similarity between them.     

Changes of genetic parameters in successive gynogenetic generations and some calculations for carp gynogenesis

Summary In artificial gynogenesis diploidization of the sperm activated zygote is achieved by retention of the second polar body and by this method perfectly normal and fully viable broods can easily be reared. Studying this form of propagation, we determined the coefficient of inbreeding (F), the fixation index (P) and the degree of genotypic identity (I) introduced for characterizing the isogeneity of gynogenetic populations with the help of the parameters of the probability distribution of meiotic recombination between the centromere and the genes.All three parameters were linear functions of the moments of the distribution of recombination probabilities. The theoretical relationships were investigated in the artificial gynogenesis of the carp.It appeared without exception that the progress of inbreeding made by gynogenesis largely depends on the distribution of the recombination probabilities of the genes. The rate of increase of the F value was more rapid only in the first few generations (2–5) compared to sibmating. The increase of the F value slowed in subsequent generations and fell significantly below the values obtained by sibmating. The degree of genotypic identity (I) is not as sensitive to the type of the distribution of recombination probability. In the case of gynogenesis, the value of I abruptly converges to l much more abruptly than in the case of sibmating.Used alone, gynogenesis does not enable the production of homozygous strains to any great extent. However, partly heterozygous, but isogenic, linea can be rapidly produced.     

Mitochondrial DNA variation in diploid and triploid forms of silver crucian carp Carassius auratus gibelio

Variation of two mtDNA fragments amplified in polymerase chain reaction was compared by the RFLP method in a population of the sympatric bisexual (diploid) and gynogenetic (triploid) forms of silver crucian carp. The mtDNA haplotypes of all individuals differed in at least 2.5% of nucleotide substitutions and fall into two phylogroups. All gynogenetic individuals have haplotypes of a single phylogroup, whereas haplotypes of diploid fish belonged to both mtDNA phylogroups. This testifies to a possibility of transformation of gynogenetic individuals into a bisexual form, whereas the reverse process is either extremely rare or impossible.     

人工雌核发育鲢的遗传多样性及异源遗传物质整入的RAPD分析

运用RAPD技术对连续二代人工雌核发育鲢的遗传多样性及异源遗传物质的整入进行了分析 ,结果表明 :一代雌核发育鲢 ,个体间遗传相似度为 0 94 5— 0 995 6 ,多样性指数为 0 175 ;二代雌核发育鲢 ,个体间遗传相似度为0 96 15— 1 0 0 ,平均为 0 985 2 ,多样性指数为 0 0 6 2。研究揭示经过连续二代人工雌核发育后 ,其遗传多样性明显减少 ,种质进一步纯化。通过对雌核发育鲢二代、亲本鲢和雄鲤的RAPD扩增比较 ,发现雌核发育鲢含有少数与父本相同的特异DNA扩增带 ,而亲本鲢没有 ,在基因水平上表明雌核发育鲢整入了雄鲤的遗传物质     

Mitochondrial DNA Variation in Diploid and Triploid Forms of Silver Crucian Carp <Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>

Variation of two mtDNA fragments amplified in polymerase chain reaction was compared by the RFLP method in a population of the sympatric bisexual (diploid) and gynogenetic (triploid) forms of silver crucian carp. The mtDNA haplotypes of all individuals was shown to be divided into two phylogenetic groups differing by at least 2.5% of nucleotide substitutions in their sequences. All gynogenetic individuals have haplotypes of a single phylogroup, whereas haplotypes of diploid fish belonged to both mtDNA phylogroups. This testifies to a possibility of transformation of gynogenetic individuals into a bisexual form, whereas the reverse process is either extremely rare or impossible.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 811–816.Original Russian Text Copyright © 2005 by Brykov, Apalikova, Eliseikina, Kovalev.     

Restriction cleavage map of mitochonrial DNA from the yeast Saccharomyces cerevisiae.

Mitochondrial DNA (mtDNA) from the yeast Saccharomyces cerevisiae was cleaved by restriction endonucleases Eco RI, Hpa I, Bam HI, Hind III, Pst I, and Sal I, yielding 10, 7, 5, 6, 1, and 1 fragments, respectively. A physical ordering of the restriction sites on yeast mtDNA has been derived. Yeast mtDNA cannot be isolated as intact molecules, and it contains nicks and gaps which complicate the use of conventional fragment mapping procedures. Nevertheless, the position of each of the restriction sites was obtained primarily by reciprocal redigestion of isolated restriction fragments. This procedure was supplemented by co-digestion of mtDNA with a multisite enzyme and a single-site enzyme (i.e., Sal I or Pst I) which provided a unique orientation for overlapping fragments cleaved by Sal I or Pst I. The data obtained from these approaches were confirmed by analysis of double and triple enzyme digests. Analysis of partial digest fragments was used for positioning of the smallest Eco RI fragment. A comparison of mtDNA from four grande strains (MH41-7B, 19d, TR3-15A, and MH32-12D) revealed similar, but slightly varying restriction patterns, with an identical genome size for each of approximately 5 X 10(-7) d or 75 kb. A fifth grande strain, D273-10B from S. cerevisiae, revealed restriction patterns different from those of the above strains, with a smaller genome size of 70 kb.     

鲫鱼的人工和天然雌核发育

用照射处理的草鱼或华南鲤的精液授精的鲫鱼卵为单倍体雌核发育;所有单倍体胚胎都是畸形,并在孵化前死亡。如果用照射精液授精的鲫鱼卵在18—19℃的水中放置2分钟,然后在0—3℃的冰水中放置20分钟,再放回室温水中孵化,可以从每百颗受精卵中得到4.5—11.9(平均8.34)尾成活的二倍体仔鱼。根据38尾银鲫(其中33尾捕自黑龙江省方正县双凤水库)的细胞学和胚胎学检查,证实它们是雌核发育繁殖的。