【作者】 陈红菊；

【导师】 谭景和； 姜运良；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 泰山赤鳞鱼(Varicorhinus sp.),是泰山的一种珍贵野生鱼种,肉质分析结果显示泰山赤鳞鱼具有极高的营养价值和特殊的药用价值。为保护这一珍贵的野生鱼种,改良其种质资源,增加其产量,本文首先对泰山赤鳞鱼卵胚胎发育进程和特征进行了观察研究,结果发现泰山赤鳞鱼卵为沉性卵,稍呈粘性,呈橙黄色或淡黄色,未吸水的卵直径为1.65 mm～1.82 mm,吸水后卵径最大达2.38 mm～2.45 mm。在水温22.5℃～24.5℃的条件下,泰山赤鳞鱼卵历时46 h 50 min完成整个胚胎发育过程,经过了胚盘形成期、卵裂期、囊胚期、原肠期、神经胚期、器官形成期、孵化期7个主要阶段。初孵仔鱼全长6 mm～6.5 mm,肌节数为46～48,具心跳、血液循环、无色素。在此基础上克隆了泰山赤鳞鱼BMP11的cDNA序列,并研究了此基因在不同胚胎发育时期及不同组织中的表达状况。从泰山赤鳞鱼的cDNA中扩增出一条643bp的条带,与斑马鱼的BMP11序列同源性高达92.34%,证明了此基因在不同物种间有高度的保守性。选择了11个不同的胚胎发育时期研究BMP11基因的表达规律,结果表明:BMP11基因在原肠晚期开始表达,虽然表达量并不恒定,但是在整个胚胎发育期都有表达,证明这是一个胚胎发育的重要基因。另外,检测BMP11基因在三个年龄的鱼(1龄,2龄,5龄)不同组织中的表达规律。研究表明:不同的年龄阶段表达存在差异,但还存在一些共同的规律:在肌肉、鳃、卵巢、脑中表达量均较高;在脾脏、肾脏、肠中都比较低。为了确定泰山赤鳞鱼的分类地位,并研究泰山赤鳞鱼与本亚科其他鱼类的进化关系,本文克隆了cytb和D-loop全序列。结果表明cytb全序列为1140bp,没有插入和缺失,其中单态位点828个,多态位点312个,占整个序列的27.37%。碱基含量平均为T 27.3%、C 29.1%、A 29.1%、G 14.5%,T+A(56.4%)明显高于G+C(43.6%)。转换明显多于颠换Ts/Tv=4.6。基于cytb序列的聚类结果表明:四种聚类树上都可明显看出泰山赤鳞鱼和多鳞铲颌鱼聚在一起,二者为同种鱼。从聚类树还可看出所研究的鲃亚科鱼类共聚为5大支系,分别属于鲃亚科的五个属:突吻鱼属、光唇鱼属、倒刺鲃属、四须鲃属和金线鲃属。在突吻鱼属内泰山赤鳞鱼和多鳞铲颌鱼先聚在一起,然后和白甲鱼聚类,二者关系很近,远远没有达到亚属的分化水平。厚唇鱼和细身光唇鱼个体之间分别聚类后又聚在一起,明显属于同属鱼类;刺鲃和倒刺鲃也是分别聚类后又聚在一起,同属于倒刺鲃属的不同亚属;金线鲃属两个种聚在一起;宽头四须鲃单独为一类,这与5属鱼类的地理分布大致吻合。泰山赤鳞鱼与鲃亚科其他属的亲缘关系为:泰山赤鳞鱼与光唇鱼属的关系最近,其次是倒刺鲃属,与四须鲃属和金线鲃属的关系都较远。基于本文的研究结果,根据分子钟推断了泰山赤鳞鱼与鲃亚科其他鱼类的分化时间。金线鲃属和泰山赤鳞鱼关系最远(15.2%～16.5%),分化时间大约是三千二百万年前;其次是四须鲃属( 14.2%～15.0 %),分化时间大约在二千九百万年前;与倒刺鲃属的分化( 13.0%～14.6% )大约在二千八百万年前;光唇鱼属和泰山赤鳞鱼关系最近(10.7%～13.8%),分化时间大约在二千四百万年前。D-loop全序列约1024bp,在序列中有一个微卫星存在,因此个体间多态性主要是微卫星核心序列重复数不同造成的。变异位点11个,占整个序列的1.74%。碱基含量平均为T 32.4%、C 20.8%、A 33.6%、G 13.3%。转换稍高于颠换Ts/Tv=1.5。根据D-loop全序列分析了泰山赤鳞鱼和多鳞铲颌鱼个体之间的遗传距离为0%～0.5%,与泰山赤鳞鱼(0.1%～0.6%)和多鳞铲颌鱼(0%～0.5%)个体之间的距离几乎一样。而与作为外群的鲮鱼之间的距离分别为:20.3%～20.8%和20.2%～20.5%,也基本上是一样的。从四种聚类图上均可看出,泰山赤鳞鱼和多鳞铲颌鱼聚在一起,两类群之间没有任何分别。本文还利用5对多态性很好的微卫星引物进行扩增,然后进行聚丙烯酰胺凝胶电泳,利用软件分析了两个群体的基因频率、哈代-温伯格平衡、遗传距离和遗传相似性,发现泰山赤鳞鱼和多鳞铲颌鱼的多态性很差,都是非常纯合的群体。两个群体遗传距离非常小,为0.0113,而遗传相似性高达0.9887。因此,无论从核外mtDNA cytb、D-loop全序列分析,还是从核内DNA微卫星标记分析均可看出泰山赤鳞鱼和多鳞铲颌鱼聚在一起,二者没有分别。更多还原

【Abstract】 The Varicorhinus sp. is a kind of precious and wild fish in mountain Tai, it’s meat have high nutritional value and special remedy value. In order to preserve this rare species, such as improving its germplasm resource and increasing its yield, we observed characteristics of Varicorhinus sp.embryos during the embryonic development process. We found that the eggs were sinkable, slightly sticky and with color of orange or yellowish. Before swelling, eggs were 1.65 mm～1.82 mm in diameter, and the diameter of swelled eggs were 2.38 mm～2.45 mm. Under 22.5℃～24.5℃of water, it took 46 hours and 50 minutes for the fertilized eggs to hatch. The embryonic development process could be divided into seven stages, including blastoderm formation, cell cleavage, blastula, gastrula, neural, organ forming and hatching stage. The total length of newly hatched larvae were 6 mm～6.5 mm and the myomeres were 46～48, and it had heartbeat and blood circulation, but no pigment in body.The sequence of BMP11 gene was cloned from Varicorhinus sp. cDNA, and its expression in different stages of embryonic development and of different tissues were examined. The Varicorhinus sp. BMP11 (643bp) has a sequence homology of 92.34% to zebrafish BMP11,which demonstrated the gene was highly conserved among different species. Eleven different stages of embryonic development were selected to study the expression regularity of BMP11, the result showed the gene was expressed during the whole stages of embryonic developing. Although its expression amount was not stable, it was an important gene for embryonic developing. In addition, the expression of BMP11 were different from ages, and the expressing quantities were more in muscles , gills, ovary and brain, less in spleen, kidney and intestines.To make sure the classification status of Varicorhinus sp. and the evolutionary relationship between Varicorhinus sp. and other fish species in the same Barbinae, we cloned mtDNA cytb and D-loop sequence. The results showed that there were 828 monophyletic sites and 312 polymorphic sites(27.37%) in the whole 1140 bp of Varicorhinus sp. cytb,without insertion and deletion. The results of homologous fragment analysis showed that the mean contents were T= 27.3%、C=29.1%、A=29.1% and G=14.5%, the content of (T+A) (56.4%) was significantly higher than that of (G+C)(43.6%). The times of transition were much higher than transversion, and the ratio between Transition and transversion was 4.6. Phylogenetic tree of cytb showed that the Varicorhinus sp. and Varicorhinus macrolepis clustered together, which demonstrated they were the same species. Based on the Phylogenetic tree of cytb,we obviously concluded that all of the fishes clustered to five branches, which belonged to five genus of Barbinae—Varossocheilus, Spinibarbus, Acrossocheilus, Barbodes and Sinocylocheilus. Firstly, Varicorhinus sp. and Varicorhinus macrolepis clustered together, then they were clustered with V.simus, which showed the relationships among them were closer than to the others. Species of A.elongatus and A.labiatus clustered firstly, then two subgenus clustered together. S.caldwelli and S.denticulatus belonged to the subgenus of Spinibarbus. The two species of Sinocylocheilus clustered together. B.laticeps was a group alone. The above results were in accord with their geographical distributions. The relationship between Varicorhinus sp. and the other genus as followed: the relationship between Varicorhinus sp. and Acrossocheilus was closest, next is Spinibarbus and Barbodes, and the relationship between Varicorhinus sp. and Sinocylocheilus was the farthest. According to the findings of this paper, we inferred the divergency time of Barbinae from the molecular evolution rate. The divergence of Sinocylocheilus (15.2%～16.5%) occurred about 32 Mya before, Barbodes(14.2%～15.0 %)occurred about 29 Mya before, Spinibarbus(13.0%～14.6%)occurred about 28 Mya before, and Acrossocheilu (10.7%～13.8%)occurred about 24 Mya before.The whole sequence of Varicorhinus sp. D-loop included 1024 bp, in which there was a microsatellite causing the polymorphism among individual, and there was 11 variation sites ( percentage 1.74%) in it. The homologous fragment analysis showed that the average content of T,C,A,G were 32.4%、20.8%、33.6%、13.3%, respectively, T+A content (66.0%)was higher than G+C(34.0%). Transition was higher lightly than transversion, and the ratio between Transition and transversion was 1.5. Based on the whole sequence of Varicorhinus sp. D-loop, we analyzed the genetic distance(0～0.5%) between Varicorhinus sp. and Varicorhinus macrolepis, which was same almostly to the genetic distance between individual of Varicorhinus sp. (0.1%～0.6%) and Varicorhinus macrolepis(0～0.5%). The genetic distance between the two species and Cirrhinus molitorella was same almostly (20.3%～20.8% and 20.2%～20.5%). Phylogenetic tree of D-loop also showed that the Varicorhinus sp. and Varicorhinus macrolepis clustered together. The results above demonstrated they were the same species.In addition,we also analyzed the gene frequency, Hardy– Weinberg equilibrium,genetic distance,genetic similarity of the two group using microsatellite marker. The result revealed the polymorphism of Varicorhinus sp. and Varicorhinus macrolepis was very poor,the genetic distance between two group was vey small (0.0113), and the genetic similarity was high (1.9887).So, we could conclude that Varicorhinus sp. and Varicorhinus macrolepis are one species by the sequence analysis of mtDNA cytb、D-loop and microsatellite marker.更多还原