【作者】 位正鹏；

【导师】 孔晓瑜；

【作者基本信息】 中国海洋大学 ， 动物学， 2008， 硕士

【摘要】 由于在双壳贝类中（M. trossulus, M. edulis,M. galloprovincialis, M. californianus,菲律宾蛤仔,罗纹贻贝（Geukensia demissa）,以及珠蚌科的Pyganodon grandis, P. fragilis,和Fusconaia flava）存在双单性遗传现象,因此本文拟初步探讨一下与紫贻贝位于同一个科的翡翠贻贝Perna viridis是否具有双单性遗传现象,并对翡翠贻贝的三个近缘种（P.viridis,P.canaliculus和P.perna）进行了初步的系统分析。应用通用引物COIL 1490和COIH 2198对翡翠股贻贝的性腺和体细胞线粒体DNA进行PCR扩增,获得661bp长度的COI基因片段,经过比对性腺与体细胞的COI片段,发现雄性性腺与体细胞COI基因均为一个单倍型,即体内只有一种线粒体DNA类型,没有发现双单性遗传现象,雌、雄性腺的COI基因片段变异率很低（0.31%）。应用PAUP构建了NJ树、MP树以及贝叶斯法构建了贝叶斯树,对股贻贝属三种间的系统关系进行了分析,结果表明,P.viridis与P.canaliculus和P.perna之间的分化与分歧年代的估算是相吻合的。香港巨牡蛎（Crassostrea hongkongensis）隶属于软体动物门（Mollusca）双壳纲（Bivalvia）珍珠贝目（Pterioida）牡蛎科（Ostreidae）巨牡蛎属（Crassostrea）,为中国南方沿海广泛分布的物种。根据太平洋牡蛎（C.gigas, NC₀01276）和美洲牡蛎（C.virginica, AY905542）全序列,用Primer Premier 5.0设计并筛选了10对引物,获得了10个片段,测序并拼接,得到了香港巨牡蛎线粒体基因组全序列,其长度为16475bp。识别了22个tRNA基因、2个rRNA基因和12个蛋白编码基因和1个主要非编码区的完整序列,除主要非编码区外,测定序列中还存在1546bp的间隔子分散在线粒体基因组中。确定了各基因在线粒体上的排列位置。与太平洋牡蛎和美洲牡蛎相比,香港巨牡蛎线粒体基因组中没有发现基因重排。计算了全序列的碱基组成,蛋白编码基因,tRNA,rRNA以及间隔子和非编码区的碱基组成,在全序列中A+T含量为65.4%,蛋白编码基因的A+T含量为64.6%,tRNA基因的A+T含量为64.2%,rRNA基因的A+T含量为60.5%,间隔子的A+T含量为72.1%,主要非编码区的A+T含量为77.8%。以轻链核酸序列为标准计算的GC-skew =-0.207,AT-skew=0.134。12个蛋白编码基因,rRNA基因以及tRNA基因均为H链编码,没有L链编码的情况。除了tRNA-His与ND4蛋白编码基因有一个碱基的重叠外,其他的基因均被tRNA基因和间隔子间隔,间隔子从1到294bp不等。分析了各基因的片段的序列特征,发现线粒体基因组中蛋白编码基因存在ATG、ATA两种常见的起始密码子,Cyt b基因的起始密码子为ATA,和美洲牡蛎以及太平洋牡蛎的略有不同。终止密码子只有TAA和T（仅有ND4L基因）两种。研究tRNA基因潜在的二级结构发现氨基酸接受臂和反密码子环在种间没有较大变异,变异主要集中在TΨC环和臂。对线粒体基因组中各基因的进化速率进行了比较。结果表明,各基因进化速率为脱氢酶亚基基因和细胞色素b基因﹥ATP酶亚基基因﹥细胞色素氧化酶亚基基因﹥tRNA基因﹥rRNA基因。在细胞色素氧化酶基因中,COIII是进化速度最快的基因,而在NADH中进化最快的则是ND2和ND6基因。Heteroconchia亚纲的菲律宾文蛤为外类群,研究了翼型亚纲牡蛎目牡蛎科巨牡蛎属的香港巨牡蛎、太平洋牡蛎和美洲牡蛎与贻贝目贻贝科的紫贻贝（M.edulis）以及Pectinoida目海扇蛤科扇贝属的海湾扇贝（Argopecten irradians）之间的系统关系。采用从单一基因到多基因结合到线粒体基因组的方式对系统关系进行多方位分析。用软件paup和MrBayes构建了MP、NJ、ML和BI系统树。在系统树中,香港巨牡蛎和太平洋牡蛎首先聚为一支,且随着基因数的增加支持率增加,用所有基因构建系统树时支持率达到100%。这表明了多基因相结合用作系统分析可以弥补单一基因由于功能、进化方式等方面的限制而带来的弊端,多基因能较全面的揭示研究种之间的系统关系。因此线粒体基因组可以比较准确的揭示物种间的系统关系,对于分类地位相差较远的物种间的系统分析,线粒体基因组是非常有用的标记。与牡蛎属现有研究结果相比,用16s rRNA基因和COI基因计算的种间遗传距离与其他研究者所计算的遗传距离基本一致。随着系统分析所用基因的增多,计算的种间遗传距离趋向于用线粒体中单一基因计算的遗传距离的平均值。因此认为基因结合适合用于属间系统关系的研究。当所有基因都用于系统分析时,种间遗传距离在0.15⁰.368之间。更多还原

【Abstract】 It is known that doubly uniparental inheritance （DUI） mitochondrial DNA are present in the species M. trossulus, M. edulis,M.galloprovincialis, M.californianus, Geukensia demissa, R. philippinarum, Pyganodon grandis, P. fragilis and Fusconaia flava. In this study, we examined whether the phenomenon of double uniparental inheritance exist in mussel Perna viridis, a species in the same family with M. galloprovincialis. We also analyzed the phylogenetic relationship of the three species from the same genus （P. viridis, P. canaliculus and P. perna） based on partial mitochondrial COI gene data.Partial mt COI gene sequences from the gonads （male and female） and somatic tissue of Perna viridis were separately amplified using the universal primers COIL 1490/COIH 2198. A 661bp nucleotide sequence of COI gene was obtained. It is found that this sequence was identical among different tissues of male individual, and low divergence （0.31%） was detected between male and female sequences. Only one haplotype was found in both female and male sequences. No doubly uniparental inheritance （DUI） was detected. Together with available COI sequences of other Perna species from GenBank, NJ tree, MP tree and Bayesian inference （BI） tree were constructed respectively with PAUP and MrBayes, and phylogenetic relationships among them were analyzed accordingly. Results showed that the sequence divergence among these 3 species was consistent with the estimation of time of differentiation.The oyster Crassostrea Hongkongensis, belongs to Mollusca-Bivalvia-Pterioida- Ostreidae-Crassostrea, is one of the most important cultured oysters along the South China Sea. Ten pairs of genus-specific primers were designed in this study based on analysis of the complete mitochondrial DNA sequences of Pacific oyster （Crassostrea gigas, NC₀01276） and eastern oyster （Crassostrea virginica, AY905542）. Ten large fragments were amplified by long PCR accordingly. Through primer walking sequencing and sequnce assembly, the complete mitochondrial sequence of 16,475bp was obtained. The genome contains 22 transfer RNA genes （tRNA）, 2 ribosomal RNA genes （rRNA）, 12 protein-coding genes, 1 dominating noncoding region, and 1562 bp noncoding inter-genic sequences. In comparison with other mitochondrial genome of Crassostrea species, no protein-coding gene rearrangement was found in C. hongkongensis.The nucleotide compositions were calculated for the complete sequence, protein-coding genes, tRNA genes, rRNA genes, Intergenic nucleotide and dominating noncoding region, respectively. The AT content of dominating noncoding region is the highest, with the value of 77.8%, and that of Intergenic nucleotide was 72.1%. The AT content of complete sequence, protein-coding genes, tRNA genes and rRNA genes are 65.4%, 64.6%, 64.2% and 60.5%, respectively. As for the criterion of L-strand, GC-skew=-0.207, AT-skew=0.134.All of the genes are encoded in H-strand. Most of the protein-coding genes are separated by tRNA genes or spacer with size range from 1bp to 294bp in length, except for tRNA-His and ND4 genes which have 1bp overlapped.In protein-coding genes, only two type of start codon are found: ATG and ATA. The start codon exist at the CytB gene in Crassostrea hongkongensis is CTA, which are CTA and TTA in the Pacific oyster and eastern oyster respectively. The start codon CTA and TTA both code for the amino acid Leu. Two type of stop codon are found, they are TAA and T（only ND4L gene for T）. In tRNA genes, anticodon loop and the amino acid arm are the most conserved regions among species.Evolutionary rate of different genes are compared. Results show that NDAH dehydrogenase subunits （ND1-ND6） and Cyt B genes are the fastest evolved genes, ATP6 subunits gene take second place, and then followed by Cytochrome c oxidase subunits gene and then are the tRNA genes. Evolutionary rate in small and large subunits rRNA genes （12s rRNA and 16s rRNA） are the lowest. In Cytochrome c oxidase subunits, COIII has a higher evolutionary rate than the other two.In NDAH dehydrogenase subunits, evolutionary rate are higher in ND6 and ND2.Phylogenetic relationship among three oyster species Crassostrea hongkongensis, C. gigas, C. virginica and M. edulis （Mytiloida: Mytilidae） and Artemia franciscana （Pectinoida: Pectinidae） are analyszed. Ruditapes philippinarum （Heteroconchia: Veneridae） is used as out-group. Every single gene, genes encode the same protein group, all protein-coding genes, all rRNA genes, all tRNA genes and all genes as one fragment are used respectively to construct NJ phylogenetic tree, MP phylogenetic tree, ML phylogenetic tree and BI phylogenetic tree by the program Paup and MrBayes. Most of the results show that C .hongkongensis is close to C. gigas, and is different from C. virginica. When single gene is used, the bootstrap supporting rate is low. When genes are used together, the bootstrap supporting rate is up to 100%. This result indicates that genes used together can complement the defect of each other’s, hence it is more resolvable in phylogenetic research. Therefore it is evident that mitochondrial genome can be used to research relationship among far-related species .Pairise distances among the three oysters calculated with COI gene and 16s rRNA gene sequence are consistent with the results from others..Pairwise distance is also estimated with different gene sequences. The values between species calculated with rRNA genes and tRNA genes are low. But it is very high when calculated by ATP subunit genes and NADH subunit genes.When different genes are combined, species with distance can be seen as different genera. When all genes are combined, the pairwise distances are ranged between 0.15 and 0.368.更多还原