三倍体湘云鲫线粒体DNA序列变异性分析

对26尾三倍体湘云鲫的线粒体tRNA-Thr基因、tRNA-Pro基因和部分控制区的核苷酸序列进行了测定,获得26条长度为837—839 bp的同源基因序列,共发现65个多态性核苷酸变异位点,多态位点比例为0.077,定义了8种单元型。在湘云鲫8种单元型中确认了DNA复制终止相关的序列TAS、中央保守区序列(CSB-F、CSB-E和CSB-D)和保守序列CSB1,8种单元型含有3—5个TAS序列。在65个变异位点中,大部分序列变异为转换,8种单元型之间的序列差异在0.1%—6.3%之间。该研究为三倍体湘云鲫的繁殖和遗传改良提供了一些有价值的信息。     

异源四倍体鲫鲤及其原始亲本遗传变异的微卫星标记分析

采用从鲤中分离出来的32对微卫星DNA标记,对异源四倍体鲫鲤、红鲫和野鲤的基因组DNA进行了研究。在筛选出的15对微卫星引物中,随引物不同,各等位基因数为1～8个,大小在100～420bp之间。从3个不同群体内部的遗传相似系数来看,异源四倍体鲫鲤个体之间的遗传相似系数最大,说明异源四倍体鲫鲤群体内部的遗传变异程度最低,已经形成了一个遗传性状稳定的群体。从3个不同群体之间的遗传相似系数来看,异源四倍体鲫鲤和红鲫遗传相似系数为0．5625,和野鲤的遗传相似系数为0．5125,说明异源四倍体鲫鲤接受原始母本的遗传物质比原始父本野鲤要多一些。微卫星标记与以前报道的RAPD标记的检测结果是相似的,然而由微卫星标记获得的种群内和种群间的遗传距离均大于RAPD,说明微卫星标记比RAPD标记显示出更高的个体多态性。     

三倍体鲫鱼--异源四倍体鲫鲤(♂)×金鱼(♀)

利用雄性四倍体鲫鲤与雌性二倍体红色双尾金鱼交配 ,制备了 1种新型三倍体鲫鱼 ,并对Ⅰ龄三倍体鲫鱼染色体数目和组型、性腺指数和性腺结构、外形、生长速度等生物学特征进行了系统研究。结果表明 :这种三倍体鲫鱼染色体数目为 3n =15 0 ,核型公式为 33m 5 1sm 33st 33t;在繁殖季节 ,三倍体鲫鱼卵巢和精巢指数明显低于作为对照的红鲫卵巢和精巢指数 ;组织学切片表明三倍体鲫鱼的卵巢和精巢不能产生成熟的卵子和精子 ,证明它们是不育的。三倍体鲫鱼体色为青灰色 ,单尾 ,具有一些介于其父母本———异源四倍体鲫鲤和红色双尾金鱼之间的外形特征 ,如具有 1对肉眼不易观察到的短须 ,侧线鳞式为 31 6 7,背鳍为 18,体高 体长之比为 0 5 ,比湘云鲫的体高 体长之比 (0 4 1)有明显的增加。经过约 8个月的饲养 ,该三倍体鲫鱼平均重量为 35 0g,最大个体达 5 5 0g。在Ⅰ龄鱼中 ,雌性三倍体鲫鱼的个体生长速度快于雄性个体。与三倍体湘云鲫相比 ,该三倍体鲫鱼保留了生长速度快、不育等优点 ,同时新添了体高 体长比值更高、肉质更甜美的特点 ,为 1种有推广前途的新型三倍体鲫鱼     

异源四倍体鲫鲤群体遗传多样性的RAPD分析

湖南师范大学和湘阴县东湖渔场合作,经过10多年的研究,获得了两性可育的异源四倍体鲫鲤群体[1—7],目前已经繁殖到了F14。异源四倍体鲫鲤具有4套染色体(4n=200),它们的遗传多样性水平怎么样?这是关系到异源四倍体鲫鲤这一新的种质资源能否生存和进化的问题,有必要从分子水平进行深入的研究。随机扩增多态DNA(Random Am-plified Polymorphic DNA,RAPD)是建立在PCR基础之上的一种DNA分子标记技术[8],具有简便、快速、实验成本低等特点,因此被广泛应用于物种的遗传多样性分析、亲缘关系的探讨和系统进化等方面的研究[9—15]。本研究采用RAPD技术,检测了异源四倍体鲫鲤群体的遗传多样性水平,以期为异源四倍体鲫鲤的人工繁殖、种群复壮以及种质资源保护提供DNA水平上的依据。     

四倍体鲫鲤、三倍体湘云鲫染色体减数分裂观察

用精巢细胞直接制片法观察了异源四倍体鲫鲤、三倍体湘云鲫和二倍体红鲫、湘江野鲤精母细胞染色体第一次减数分裂中期配对情况 ;作为对照 ,观察了上述四种鱼肾细胞的有丝分裂中期染色体。在精母细胞第一次减数分裂中 ,异源四倍体鲫鲤同源染色体两两配对 ,形成 10 0个二价体 ,没有观察到单价体、三价体和四价体 ;三倍体湘云鲫精母细胞形成 5 0个二价体和 5 0个单价体 ;红鲫和湘江野鲤精母细胞分别形成 5 0个二价体。肾细胞检测表明异源四倍体的染色体数目为 4n =2 0 0 ;湘云鲫为 3n =15 0 ;红鲫和湘江野鲤分别为 2n =10 0。减数分裂时染色体分布情况与肾细胞染色体检测结果相吻合。具有四套染色体的异源四倍体鲫鲤在减数分裂中只形成 10 0个二价体 ,而不形成 2 5个四价体或其它形式 ,为产生稳定一致的二倍体配子提供了重要的遗传保障 ,也为人工培育的异源四倍体鲫鲤群体能够世世代代自身繁衍下去提供了重要的遗传学证据。三倍体湘云鲫在减数分裂过程中出现二价体、单价体共存 ,同源染色体在配对和分离中出现紊乱 ,导致非整倍体生殖细胞的产生 ,为湘云鲫的不育性提供了染色体水平上的证据     

三倍体湘云鲫3个Sox基因保守区的序列分析

SOX/Sox基因家族成员的主要特征是具有一个HMG(high mobility group)保守盒,其编码的蛋白质可以和DNA序列特异性结合,是一类重要的转录调控因子[1],在个体发育过程中SOX/Sox基因对性别分化和组织器官形成起着重要作用。SOX/Sox基因的研究不仅可以揭示性别分化和胚胎发育的机制,而且可为某些遗传疾病的诊断和治疗提供分子方面的资料,因而有着非常重要的理论和应用价值。     

异源四倍体鲫鲤雌核发育后代及其亲本RAPD分析

异源四倍体鲫鲤是湖南师范大学鱼类发育生物学实验室和湖南湘阴县东湖渔场在红鲫(♀)和湘江野鲤(♂)的杂交后代中选育出来的四倍体鱼,目前已连续繁殖14代(F3-F16),已形成一个四倍体性能代代相传、遗传性状稳定的四倍体鱼群体,这是世界上唯一人工培育的两性可育的异源四倍体鱼[1—2]。利用四倍体鱼与二倍体白鲫、二倍体鲤鱼杂交,可获得生长快、肉质鲜美、抗病力强等优良性状的不育三倍体湘云鲫、三倍体湘云鲤[3],并已在全国28个省市推广养殖,取得了显著的经济和社会效益。异源四倍体鲫鲤雌性个体产生的二倍体卵子具有两套染色体,在没有染色…     

异源四倍体鲫鲤雌核发育后代的RAPD分析

在优化RAPD(随机扩增多态性DNA)检测条件基础上,从134个随机引物中筛选出53个扩增较好且多态性强的引物,对异源四倍体鲫鲤第1代(G1)、第2代(G2)人工诱导的雌核发育二倍体后代群体的DNA多态性及分子标记进行了分析。结果显示,53个随机引物在G1群体和G2群体中检测到的位点数分别为541、511,其中多态性位点数分别为70、52,多态位点比例分别为12.94%、10.18%。两个群体的平均遗传距离分别为0.0732、0.0464。研究表明,经过连续2代人工雌核发育,G2的遗传多样性明显减少,种质进一步纯化。还从53个随机引物的扩增谱带中找到了2个引物(S50、S223)的特异扩增谱带,可以作为第1、2代雌核发育群体间的分子遗传标记。由计算机软件程序构建的分支系统树清晰地反映了两个雌核发育群体及其个体间的相互关系。       

异源四倍体鲫鲤雌雄差异的RAPD标记

异源四倍体鲫鲤是从红鲫和湘江野鲤的杂交后代选育出来的,已经形成了一个遗传性状稳定的四倍体鱼新种群。用异源四倍体鲫鲤(雄性)和二倍体白鲫(雌性)生产的三倍体湘云鲫已经在全国推广应用。因此,如果能够了解异源四倍体鲫鲤的性别分化机制,人为地控制异源四倍体鲫鲤的性别分化,生产出大量的超雄鱼,这对于三倍体湘云鲫的产业化生产有重要的意义。刘少军等对异源四倍体鲫鲤的染色体组型进行了分析,并没有发现异源四倍体鲫鲤有明显的特化的性染色体,这说明通过细胞遗传学研究异源四倍体鲫鲤的性别遗传机制是有困难的。     

异源四倍体鲫鲤及其原始父母本细胞周期蛋白B基因克隆与分子遗传分析

细胞周期蛋白(cyclin)B是真核细胞周期运转中调控G2期至M期转化的关键因子.本实验根据斑马鱼细胞周期蛋白 B1基因的剪切方式,设计3对特异于金鱼和银鲫细胞周期蛋白B基因外显子区兼并引物,首次扩增出异源四倍体鲫鲤及其原始亲本红鲫和鲤鱼细胞周期蛋白B基因2条大小分别约为2.4 kb和2.1 kb的片段.测序及比对分析表明：这3种鱼的细胞周期蛋白B基因2个片段均包含8个外显子和7个内含子.内含子剪切位点符合GT/AG规则,推测细胞周期蛋白B基因2个片段可能是细胞周期蛋白B基因的2种存在形式.异源四倍体鲫鲤与其原始父母本细胞周期蛋白B基因片段序列的比较结果表明：无论是外显子区还是内含子区,异源四倍体鲫鲤与其原始亲本都具有较高的遗传相似性,在1 025 bp的外显子序列中相同的碱基位点数达963个,为异源四倍体鲫鲤来源于红鲫和鲤鱼提供了分子证据;同时,异源四倍体鲫鲤与其原始亲本差异碱基位点的存在又表明这一独特的多倍体物种与其原始亲本存在着进化上的变异.此外,还分别以细胞周期蛋白B基因外显子和内含子序列构建了包括异源四倍体鲫鲤及其原始父母本在内的系统进化树.结果初步表明：对于亲缘关系较近的物种,用外显子和内含子序列构建的系统进化树与传统的物种进化树一致;而对于亲缘关系较远的物种,用内含子序列构建的进化树与传统的物种进化顺序存在较大差异.     

Comparative and Evolutionary Analysis of the Cytochrome b Sequences in Cyprinids with Different Ploidy Levels Derived from Crosses

The mitochondrial cyt b genes in the allotetraploid and triploid crucian carp as well as triploid common carp were isolated and completely sequenced. Their DNA sequences were compared with those derived from the cyt b genes of the red crucian carp, Japanese crucian carp, and common carp with MEGA 1.0 software. Phylogenetic analysis revealed the sister relationships between allotetraploid and diploid red crucian carp, between the triploid crucian carp and diploid Japanese crucian carp, and between triploid common carp and diploid common carp. Our results indicated the cyt b genes in the allotetraploid, triploid crucian carp, and triploid common carp were maternally inherited. Through maternal inheritance, the cyt b gene in the F11 tetraploid displayed extremely high similarity to that in the female parent red crucian carp after 11 generations (from F1 to F11 hybrids). Since the establishment of the new tetraploid stocks has great significance in analyzing evolutionary theory of vertebrate and in improving aquaculture industry, analysis of the cyt b gene and the elucidation of the variation of the cyt b gene DNA in different cyprinids prove that cyt b is a useful genetic marker to monitor the variations in the progeny of the crosses.     

The cloning of Dmc1 cDNAs and a comparative study of its expression in different ploidy cyprinid fishes

Dmc1 (disrupted meiotic cDNA) is a functionally specific gene, which was firstly discovered in yeast and then found to encode a protein required for homologous chromosome synapsis during the process of meiosis. In this investigation, we cloned the partial cDNAs of Dmc1 of diploid red crucian carp, Japanese crucian carp, common carp, triploid crucian carp and allotetraploid hybrids by using a pair of degenerate primers based on the conservative sequence of amino acids of the DMC1 protein in yeast, mouse and human. The full length cDNAs were then obtained by rapid amplification of cDNA ends (RACE). Our data showed that the full length cDNAs of Dmc1 in the three diploid fishes are all 1375 bp long, while it is 1383 bp long in triploids and 1379 bp long in allotetraploids. And despite of the variation in length, all the cDNAs encode a protein of 342 amino acids. A high homology of 97.3% of the DMC1 protein can be drawn by comparing the amino acid sequences in the three diploids, which is also of 86%, 86% and 95% similarity to human, mouse and zebrafish, respectively. A comparative study of the expression pattern of Dmc1 was carried out by RT-PCR using specific primers against the same sequences of coding regions in different ploidy cyprinid fishes, from which it was showed that Dmc1 was expressed only in gonads of these five kinds of fishes. The expression pattern of Dmc1 in both ovaries and testes from different ploidy fishes within breeding season was also studied by Real-time PCR, and the results showed that the expression of this gene was greatly different among the three different ploidy fishes, which was the highest of triploid and lowest of allotetraploids. The histological sections data showed matured gonads of both diploid red crucian carp and allotetraploids in breeding season, although the latter demonstrated a higher maturation, and no gonadal maturation could be observed in triploids. In conclusion, we suggest that Dmc1 is specifically expressed in the period of meiosis in all the ploidy cyprinid fishes and directly related with the development of gonad in a manner of ploidy-independent way. And further, the high expression of Dmc1 in female triploids might be associated with abnormal meiosis and sterility.     

Comparative studies on histological and ultra-structure of the pituitary of different ploidy level fishes

The histological and ultra-structure of the pituitary in diploid red crucian carp (Carassius auratus red var.), triploid crucian carp and allotetraploid hybrids within and after the breeding season were comparatively studied. The result showed that there were six endocrine cell types in the pituitary of these three kinds of fishes, and there was an obvious difference in cell size among different ploidy level fishes. As for the same type of pituitary cells, the cell size was increased gradually with the increasing ploidy level. In the breeding season, the allotetraploid hybrids had higher proportion of gonadotropin cells (GTH) than triploids, and the trploids had higher proportion of GTH than diploids. The results were related to the earlier sexual maturity of allotetraploid hybrids and sterility of triploid crucian carp. On the other hand, among the three kinds of fishes, the proportion of somatotropin (STH) cells in triploids crucian carp was the highest, whereas that in allotetraploid hybrids was the lowest. The results might be connected with the faster growth rate of triploids and slower growth rate of allotetraploid hybrids. In addition, in GTH cells of meso-adenohypophysis after the breeding season, there were many endocrine particles in triploids, while those endocrine particles were released from the cells in allotetraploids and diploids. This result showed that the sterility of triploid crucian carp might be related to the hormone which was not released from the GTH cells. In a word, the present study indicated that the differences in the structure of pituitary among different ploidy level fishes contributed to their difference in the growth rate and gonadal development.     

The cloning of Dmc1 cDNAs and a comparative study of its expression in different ploidy cyprinid fishes

Dmc1 (disrupted meiotic cDNA) is a functionally specific gene, which was firstly discovered in yeast and then found to encode a protein required for homologous chromosome synapsis during the process of meiosis. In this investigation, we cloned the partial cDNAs of Dmc1 of diploid red crucian carp, Japanese crucian carp, common carp, triploid crucian carp and allotetraploid hybrids by using a pair of degenerate primers based on the conservative sequence of amino acids of the DMC1 protein in yeast, mouse and human. The full length cDNAs were then obtained by rapid amplification of cDNA ends (RACE). Our data showed that the full length cDNAs of Dmc1 in the three diploid fishes are all 1375 bp long, while it is 1383 bp long in triploids and 1379 bp long in allotetraploids. And despite of the variation in length, all the cDNAs encode a protein of 342 amino acids. A high homology of 97.3% of the DMC1 protein can be drawn by comparing the amino acid sequences in the three diploids, which is also of 86%, 86% and 95% similarity to human, mouse and zebrafish, respectively. A comparative study of the expression pattern of Dmc1 was carried out by RT-PCR using specific primers against the same se-quences of coding regions in different ploidy cyprinid fishes, from which it was showed that Dmc1 was expressed only in gonads of these five kinds of fishes. The expression pattern of Dmc1 in both ovaries and testes from different ploidy fishes within breeding season was also studied by Real-time PCR, and the results showed that the expression of this gene was greatly different among the three different ploidy fishes, which was the highest of triploid and lowest of allotetraploids. The histological sections data showed matured gonads of both diploid red crucian carp and allotetraploids in breeding season, although the latter demonstrated a higher maturation, and no gonadal maturation could be observed in triploids. In conclusion, we suggest that Dmc1 is specifically expressed in the period of meiosis in all the ploidy cyprinid fishes and directly related with the development of gonad in a manner of ploidy-independent way. And further, the high expression of Dmc1 in female triploids might be associ-ated with abnormal meiosis and sterility.     

Comparative studies on histological and ultra-structure of the pituitary of different ploidy level fishes

The histological and ultra-structure of the pituitary in diploid red crucian carp(Carassius auratus red var.),triploid crucian carp and allotetraploid hybrids within and after the breeding season were comparatively studied.The result showed that there were six endocrine cell types in the pituitary of these three kinds of fishes,and there was an obvious difference in cell size among different ploidy level fishes.As for the same type of pituitary cells,the cell size was increased gradually with the in- creasing ploidy level.In the breeding season,the allotetraploid hybrids had higher proportion of go- nadotropin cells(GTH)than triploids,and the triploids had higher proportion of GTH than diploids.The results were related to the earlier sexual maturity of allotetraploid hybrids and sterility of triploid cru- cian carp.On the other hand,among the three kinds of fishes,the proportion of somatotropin(STH) cells in triploids crucian carp was the highest,whereas that in allotetraploid hybrids was the lowest. The results might be connected with the faster growth rate of triploids and slower growth rate of al- lotetraploid hybrids.In addition,in GTH cells of meso-adenohypophysis after the breeding season, there were many endocrine particles in triploids,while those endocrine particles were released from the cells in allotetraploids and diploids.This result showed that the sterility of triploid crucian carp might be related to the hormone which was not released from the GTH cells.In a word,the present study indicated that the differences in the structure of pituitary among different ploidy level fishes contributed to their difference in the growth rate and gonadal development.     

从12S rRNA基因第三结构域序列分析探讨蜘蛛若干重要类群的亲缘关系

本文将12S rRNA基因序列分析应用于研究若干重要蜘蛛类群的系统关系,以对传统的分类研究结论进行验证和补充,并且探讨12S rRNA基因序列分析在蜘蛛系统发生研究中的适用性。根据12S rRNA基因第3结构域构建的分子系统树得出结论:1.圆网类(即妖面蛛总科与园蛛总科)并非单系;2.隙蛛与暗蛛较漏斗蛛具有更近的亲缘关系;3.壁钱和拟壁钱并不近缘;4.有筛器类蜘蛛为复系类群;5.12S rRNA基因第3结构域片段对推断近缘科属间的系统发生关系是有效的遗传标记。     

从12S rRNA基因片段序列研究20种蛇的系统发生关系

本文测定了中国产蛇亚目20种蛇约800bp的mtDNA 12S rDNA基因片段序列。所测序列与楔齿蜥的同源序列一起经Clustal X1.8软件比对,共有881个位点,其中变异位点有494个。以楔齿蜥为外群,用NJ法构建了4科20种蛇的进化关系树,对这20种蛇的系统发生关系作了初步探讨。研究结果表明：所研究的4个科20种蛇分成4个支系。第一个支系包括蟒科的东方沙蟒和蟒2种蛇;第二个支系为蝰科3种蛇,即草原蝰、尖吻蝮、竹叶青组成一个单系群;眼镜蛇科的眼镜蛇和银环蛇构成第三个支系;游蛇科的13种蛇构成了第四个支系,其中灰鼠蛇、乌梢蛇和赤链蛇组成一个支系,锦蛇属的7种蛇组成一个单系群,然后它们与前一支系相聚。剩下的颈槽蛇属两种聚类后与赤链华游蛇构成一个支系,并与游蛇科其它蛇组成姐妹群。第四支系首先与第三支系眼镜蛇科聚类,第二支系蝰科构成了三、四支系眼镜蛇科和游蛇科的姐妹群,第一支系蟒科在系统树的最基部,为四个科中较原始的类群。     

从1 2S rRNA基因序列探讨8种鳄类的系统学关系

测得扬子鳄（Alligator sinensis）和暹罗鳄（Crocodylus siamensis）,的mtDNA12SrRNA基因片段的部分序列,与GenBank中的2种鳄及文献中4种鳄的12SrRNA基因相应片段,经比对后构建系统树。其结果显示,现存鳄类为单系起源,可划分为3个科,即：鳄科、食鱼鳄科和假食鱼鳄科。食鱼鳄与假食鱼鳄亲缘关系较近,支持将假食鱼鳄作为食鱼鳄科的一个属,与以形态学为基础的研究结果不同。在钝吻鳄科中,扬子鳄与凯门鳄亲缘关系较远,而与密西西比鳄的亲缘关系虽较近,但它们的确存在很多差异。两者12SrRNA基因序列差异达12．12％,碱基的颠换数为9。在基于碱基转换和颠换的NJ系统树及仅基于碱基颠换的NJ系统树中,前者支持扬子鳄与密河鳄间的亲缘关系较近,而后者支持扬子鳄与凯门鳄间的亲缘关系较近,说明扬子鳄与密河鳄间亲缘关系是值得研究的问题。因本文仅根据12SrRNA基因部分序列来进行分析的,尚需进一步研究。     

人工诱导异源四倍体和倍间三倍体白鲫的红细胞观察及其相对DNA含量测定

染色体核型分析表明,异源四倍体白鲫含有两套白鲫染色体和两套红鲫染色体,新四倍体白鲫含有三套白鲫染色体和一套红鲫染色体,倍间三倍体白鲫含有二套白鲫染色体和一套红鲫染色体。为进一步阐明人工诱导多倍体的染色体倍性与细胞和细胞核及其相对DNA含量之间的相关关系,作者测量了异源四倍体、新四倍体和借间三倍体鱼红细胞及其核的大小,同时测定了它们的红细胞相对DNA含量,并与它们亲本红细胞及其精子细胞相对DNA含量进行了比较。