异源四倍体鲫鲤、三倍体湘云鲫和它们父母本线粒体DNA 12S rRNA基因遗传变异的分析

采用质粒克隆测序方法,获得了异源四倍体鲫鲤5个个体、异源四倍体鲫鲤雌核发育二倍体后代2个个体、三倍体湘云鲫2个个体及红鲫、湘江野鲤和日本白鲫各1个个体的线粒体DNA 12S rRNA基因的全序列。经对比发现,异源四倍体5个个体共享2种单元型,异源四倍体鲫鲤雌核发育二倍体后代2个个体、三倍体湘云鲫2个个体以及红鲫、湘江野鲤和日本白鲫各1个个体分别共享1种单元型。用MEGA 1.0 软件分析了它们的碱基组成和核苷酸序列差异,用邻接法构建系统进化树。它们间的序列同源性在95%~99%之间,异源四倍体鲫鲤、三倍体湘云鲫和它们母本（分别为红鲫和日本白鲫）之间的序列同源性大于异源四倍体鲫鲤、三倍体湘云鲫和它们父本（分别为湘江野鲤和异源四倍体鲫鲤）之间的序列同源性,结果表明：异源四倍体鲫鲤和三倍体湘云鲫在线粒体DNA 12S rRNA基因上具有母性遗传特征。本研究另一值得注意地方的是异源四倍体鲫鲤经过9代(F3-F11)繁殖后,在5个个体中发现了2种单元型,说明在四倍体基因库中存在遗传多样性,为四倍体基因库的繁殖、保护和种群复壮提供了一些有价值的信息。     

从ATPase8-6基因研究杂交多倍体鱼线粒体母性遗传

异源四倍体鲫鲤是世界上首例人工培育的两性可育并形成群体的且能自然繁殖的四倍体鱼。本文采用质粒克隆测序法测定了红鲫、异源四倍体鲫鲤、三倍体湘云鲫和三倍体湘云鲤的ATPase8和ATPase6基因全序列 ,结合鲤鱼、日本白鲫和斑马鱼的同源序列 ,对不同倍性水平鲤科鱼类的ATPase8和ATPase6基因进行了比较 ,分析了碱基组成、变异情况以及核苷酸和氨基酸序列差异。红鲫、鲤鱼、异源四倍体鲫鲤、日本白鲫、三倍体湘云鲫和三倍体湘云鲤之间的序列差异为 0 0 % - 1 3 4 % ,它们与外群斑马鱼之间的序列差异为 2 7 9% -31 0 %。用MEGA软件中的MP法、ME法、NJ法和UPGMA法构建分子系统树 ,得到了相似的拓扑结构。结果分析表明 ,人工杂交多倍体异源四倍体鲫鲤、三倍体湘云鲫和三倍体湘云鲤在线粒体ATPase8和ATPase6基因上具有严格的母性遗传特征。值得注意的是 ,异源四倍体鲫鲤经过 1 1代的繁育后 ,与其原始母本红鲫仍然保持了非常高的同源性 ,说明了新的异源四倍体基因库在线粒体ATPase8和ATPase6基因上拥有稳定的遗传特性。对不同倍性鲤科鱼类线粒体ATPase8和ATPase6基因的研究表明 ,ATPase8和ATPase6基因是杂交鱼后代遗传变异研究的一个很好的分子标记     

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。       

蛋白磷酸酶-2Ac在不同倍性鱼6种组织中的分化表达模式

蛋白磷酸酶-2A是最重要的丝氨酸/苏氨酸蛋白磷酸酶之一,对于调控多细胞的生命活动起着非常重要的作用.以异源四倍体鲫鲤及其二倍体父/母本(湘江野鲤/红鲫)和子代三倍体湘云鲫等为实验材料,运用Westernblot技术及荧光免疫组织化学技术等实验手段,得到了Protein PJhosphatase-2A(PP2A)的催化亚基在上述不同倍性鱼体内6种不同组织的表达模式:Protein Phosphatase-2Ac(PP2Ac)在异源四倍体鲫鲤及其二倍体父/母本及子代三倍体湘云鲫不同组织中蛋白水平均有表达,而且出现了明显的种属特异性和组织特异性,如在大脑、肌肉、肝脏三组织中,三倍体湘云鲫中PP2Ae的表达相对最高.而在肾脏组织中,PP2Ac在异源四倍体鲫鲤中的表达水平最高,父本与三倍体湘云鲫中的表达比较相近,且最低;而在性腺组织中则是父本精巢中的表达最高;在心脏组织中,PP2Ae在母本红鲫中的表达相对较高.这种明显的种属之间组织特异性可能说明了子代与父母本之间的变异性.荧光免疫组化实验结果显示,从整体水平来看,4种不同鱼的同一组织中,PP2Ac的相对定位是非常相似的,这可能说明了异源四倍体鲫鲤与其二倍体父/母本及子代三倍体湘云鲫之间的遗传相似性.研究结果为进一步探索PP2Ac在脊椎动物不同组织中的功能提供了实验依据.     

不同倍性鱼垂体细胞和超微结构比较

对繁殖季节和繁殖季节后的二倍体红鲫(Carassius auratus red var.)、三倍体湘云鲫以及四倍体鲫鲤脑垂体细胞的显微和超微结构以及组织化学特性进行了比较研究. 结果表明, 3种鱼脑垂体中都存在6种不同类型分泌细胞, 但是不同倍性鱼的垂体细胞大小存在明显差异, 就同一类细胞体积而言, 四倍体鱼垂体细胞大于三倍体垂体细胞, 三倍体垂体细胞大于二倍体垂体细胞. 在繁殖季节, 四倍体鲫鲤中腺垂体的GTH细胞所占比例最大, 其次是二倍体红鲫, 最少的是三倍体湘云鲫, 该现象与四倍体鲫鲤的性腺发育提前、三倍体湘云鲫不育有关联; 另一方面, 三倍体湘云鲫中腺垂体中STH细胞所占比例最高, 其次是二倍体, 最少的是四倍体鲫鲤, 这与三倍体湘云鲫生长速度最快、四倍体鲫鲤生长速度相对缓慢有关. 另外, 三倍体湘云鲫中腺垂体GTH细胞中的分泌颗粒和分泌小球在繁殖季节没有大量排出, 而四倍体鲫鲤和二倍体红鲫明显有大量排出, 说明三倍体湘云鲫的不育性与GTH中的激素不排出有关. 以上结果说明, 不同倍性鱼类在垂体结构方面表现出的差异与它们的生长速度、性腺发育等方面具有关联性.     

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     

二倍体鲫鲤F2产生不同倍性卵子的证据

在检测到鲫鲤F2产生3种不同大小(直径分别为0.13 cm,0.17cm和0.2 cm)类型的卵子基础上,进行了F2(♀)×红鲫(♂)及F2(♀)×四倍体鲫鲤(♂)的交配实验.通过染色体计数和流式细胞仪分析,在F2(♀)×红鲫(♂)后代中获得了四倍体、三倍体、二倍体鱼;在F2(♀)×四倍体鲫鲤(♂)后代中获得了四倍体和三倍体鱼.这两个交配组合后代中出现的不同倍性的鱼类为证明鲫鲤F2能产生三倍体、二倍体和单倍体卵子提供了进一步证据.F2(♀)×红鲫(♂)中雄性四倍体鱼的存在说明在四倍体后代中存在基因型为XXXY的个体.对上述两个交配组合后代的四倍体鱼和三倍体鱼的性腺结构观察表明四倍体鱼是可育的,而三倍体鱼是不育的.作者认为鲫鲤F2能够产生二倍体和三倍体卵子与核内复制机制和生殖细胞的融合有关.     

转青鱼生长激素基因异源四倍体鲫鲤

生态安全性是转基因鱼走向市场的瓶颈,通过转基因四倍体鱼同转基因二倍体鱼杂交获得不育的转基因三倍体鱼是解决该问题的有效途径之一.本研究构建了青鱼β-actin基因启动子和青鱼生长激素（GH）基因精确连接的＂全鱼＂基因pbcAbcGHc;并采用显微注射法将pbcAbcGHc导入异源四倍体鲫鲤受精卵.对照养殖结果表明,150日龄的转基因异源四倍体鲫鲤原代（P0）的体重及体长明显大于对照组.选择60尾P0代转基因异源四倍体鲫鲤,采用PCR方法检测出外源青鱼GH基因在P0代转基因四倍体尾鳍基因组DNA中的整合率为90%;对20尾雄性P0代转基因四倍体精液样本的PCR检测发现,13个样本具有外源青鱼GH基因的整合.在一尾生长速度显著的P0代转基因四倍体鲫鲤的肌肉、肝脏、肾脏和卵巢组织中可检测到外源青鱼GH基因的转录.本研究成功获得了具有明显生长优势的P0代转青鱼GH基因异源四倍体鲫鲤,为建立转青鱼GH基因异源四倍体鲫鲤纯系和研制不育的转基因三倍体鱼奠定了基础.     

不同倍性鱼肌间骨的比较分析

采用常规测量法和解剖法对野生鲫(Carassius auratus,2n=100)、彭泽鲫(Carassius auratus variety pengze,3n=162)、改良三倍体鲫鱼(Triploid crucian carp,即湘云鲫2号,3n=150)及其亲本改良二倍体红鲫(Carassius auratus red var,改良红鲫,♀,2n=100)和改良异源四倍体鲫鲤(Allotetreploid hybrid,四倍体鲫鲤,♂,4n=200)5种不同倍性鱼肌间骨的数目、形态和分布进行研究.结果显示,野生鲫肌间骨数目在78~83之间,平均值为81根,彭泽鲫肌间骨数目在80~86之间,平均值为84,四倍体鲫鲤肌间骨数目在77~84之间,平均值为82,但是湘云鲫2号和改良红鲫的肌间骨数目比较少,湘云鲫2号在77~82之间,平均值为79,改良红鲫在58~77之间,平均值为71.考虑到不同倍性的鱼体大小和肌节数目的不同,进一步统计了每一肌节的平均肌间骨数目,野生鲫最多(0.721),彭泽鲫次之(0.673),改良红鲫最少(0.608),湘云鲫2号次之(0.633),四倍体鲫鲤位于中间(0.653),除了两组湘云鲫2号与四倍体鲫鲤、四倍体鲫鲤和彭泽鲫外,两两间都存在显著差异.5种鱼的各种肌间骨有"I"形、"卜"形、"Y"形、一端多叉形、两端两分叉形、两端多叉形和树枝形7种类型.肌间小骨越靠前端,形态越复杂.每条鱼左右两侧的肌间骨数目不完全相等,但总体上两侧肌间骨的数目接近.在保持鱼的营养、形态和活动等基本生理功能的前提下,湘云鲫2号的肌间刺数目比野生鲫和彭泽鲫都少,所以它的食用价值较高.本研究结果说明,改良二倍体红鲫和改良三倍体鲫鱼等人工培育的杂交鱼比野生鲫的肌间刺少,为鱼类骨骼发育生物学和鱼类遗传育种提供了形态学基础.     

On the origin of the Hirudinea and the demise of the Oligochaeta

The phylogenetic relationships of the Clitellata were investigated with a data set of published and new complete 18S rRNA gene sequences of 51 species representing 41 families. Sequences were aligned on the basis of a secondary structure model and analysed with maximum parsimony and maximum likelihood. In contrast to the latter method, parsimony did not recover the monophyly of Clitellata. However, a close scrutiny of the data suggested a spurious attraction between some polychaetes and clitellates. As a rule, molecular trees are closely aligned with morphology-based phylogenies. Acanthobdellida and Euhirudinea were reconciled in their traditional Hirudinea clade and were included in the Oligochaeta with the Branchiobdellida via the Lumbriculidae as a possible link between the two assemblages. While the 18S gene yielded a meaningful historical signal for determining relationships within clitellates, the exact position of Hirudinea and Branchiobdellida within oligochaetes remained unresolved. The lack of phylogenetic signal is interpreted as evidence for a rapid radiation of these taxa. The placement of Clitellata within the Polychaeta remained unresolved. The biological reality of polytomies within annelids is suggested and supports the hypothesis of an extremely ancient radiation of polychaetes and emergence of clitellates.     

On the ontogeny of the haptor and the evolution of the Monogenea

Data on the ontogeny of the posterior haptor of monogeneans were obtained from more than 150 publications and summarised. These data were plotted into diagrams showing evolutionary capacity levels based on the theory of a progressive evolution of marginal hooks, anchors and other attachment components of the posterior haptor in the Monogenea (Malmberg, 1986). 5 + 5 unhinged marginal hooks are assumed to be the most primitive monogenean haptoral condition. Thus the diagrams were founded on a 5 + 5 unhinged marginal hook evolutionary capacity level, and the evolutionary capacity levels of anchors and other haptoral attachement components were arranged according to haptoral ontogenetical sequences. In the final plotting diagram data on hosts, type of spermatozoa, oncomiracidial ciliation, sensilla pattern and protonephridial systems were also included. In this way a number of correlations were revealed. Thus, for example, the number of 5 + 5 marginal hooks correlates with the most primitive monogenean type of spermatozoon and with few sensillae, many ciliated cells and a simple protonephridial system in the oncomiracidium. On the basis of the reviewed data it is concluded that the ancient monogeneans with 5 + 5 unhinged marginal hooks were divided into two main lines, one retaining unhinged marginal hooks and the other evolving hinged marginal hooks. Both main lines have recent representatives at different marginal hook evolutionary capacity levels, i.e. monogeneans retaining a haptor with only marginal hooks. For the main line with hinged marginal hooks the name Articulon-choinea n. subclass is proposed. Members with 8 + 8 hinged marginal hooks only are here called Proanchorea n. superord. Monogeneans with unhinged marginal hooks only are here called Ananchorea n. superord. and three new families are erected for its recent members: Anonchohapteridae n. fam., Acolpentronidae n. fam. and Anacanthoridae n. fam. (with 7 + 7, 8 + 8 and 9 + 9 unhinged marginal hooks, respectively). Except for the families of Articulonchoinea (e.g. Acanthocotylidae, Gyrodactylidae, Tetraonchoididae) Bychowsky's (1957) division of the Monogenea into the Oligonchoinea and Polyonchoinea fits the proposed scheme, i.e. monogeneans with unhinged marginal hooks form one old group, the Oligonchoinea, which have 5 + 5 unhinged marginal hooks, and the other group form the Polyonchoinea, which (with the exception of the Hexabothriidae) has a greater number (7 + 7, 8 + 8 or 9 + 9) of unhinged marginal hooks. It is proposed that both these names, Oligonchoinea (sensu mihi) and Polyonchoinea (sensu mihi), will be retained on one side and Articulonchoinea placed on the other side, which reflects the early monogenean evolution. Except for the members of Ananchorea [Polyonchoinea], all members of the Oligonchoinea and Polyonchoinea have anchors, which imply that they are further evolved, i.e. have passed the 5 + 5 marginal hook evolutionary capacity level (Malmberg, 1986). There are two main types of anchors in the Monogenea: haptoral anchors, with anlages appearing in the haptor, and peduncular anchors, with anlages in the peduncle. There are two types of haptoral anchors: peripheral haptoral anchors, ontogenetically the oldest, and central haptoral anchors. Peduncular anchors, in turn, are ontogenetically younger than peripheral haptoral anchors. There may be two pairs of peduncular anchors: medial peduncular anchors, ontogentically the oldest, and lateral peduncular anchors. Only peduncular (not haptoral) anchors have anchor bars. Monogeneans with haptoral anchors are here called Mediohaptanchorea n. superord. and Laterohaptanchorea n. superord. or haptanchoreans. All oligonchoineans and the oldest polyonchoineans are haptanchoreans. Certain members of Calceostomatidae [Polyonchoinea] are the only monogeneans with both (peripheral) haptoral and peduncular anchors (one pair). These monogeneans are here called Mixanchorea n. superord. Polyonchoineans with peduncular anchors and unhinged marginal hooks are here called the Pedunculanchorea n. superord. The most primitive pedunculanchoreans have only one pair of peduncular anchors with an anchor bar, while the most advanced have both medial and lateral peduncular anchors; each pair having an anchor bar. Certain families of the Articulonchoinea, the Anchorea n. superord., also have peduncular anchors (parallel evolution): only one family, the Sundanonchidae n. fam., has both medial and lateral peduncular anchors, each anchor pair with an anchor bar. Evolutionary lines from different monogenean evolutionary capacity levels are discussed and a new system of classification for the Monogenea is proposed.In agreeing to publish this article, I recognise that its contents are controversial and contrary to generally accepted views on monogenean systematics and evolution. I have anticipated a reaction to the article by inviting senior workers in the field to comment upon it: their views will be reported in a future issue of this journal. EditorIn agreeing to publish this article, I recognise that its contents are controversial and contrary to generally accepted views on monogenean systematics and evolution. I have anticipated a reaction to the article by inviting senior workers in the field to comment upon it: their views will be reported in a future issue of this journal. Editor