【作者】 郑将臣；

【导师】 赵金良； 程起群；

【作者基本信息】 上海海洋大学 ， 动物遗传育种与繁殖， 2011， 硕士

【摘要】 本论文基于线粒体12S核糖体RNA(mitochondrial 12S ribosomal RNA,12SrRNA)、线粒体16S核糖体RNA(mitochondrial 16S ribosomal RNA,16SrRNA)以及两个核基因:重组激活基因(Recombination activating genes,Rags)和RNA指纹蛋白35(RNA fingerprint protein 35,R35)的序列,初步分析部分龟鳖类的分子系统学特征,探讨其种间、属间、科间的分类地位。1)12SrRNA:测定了15种龟类线粒体12S rRNA基因片段的序列。比对后获得一致序列长为433bp,有191个变异位点,总变异率为43.2%,其中简约信息位点108个。A、T、G、C的平均含量分别为21.46%、34.42%、25.85%和18.27%。A+T含量(55.9%)高于G+C含量(44.2%)。在433个核苷酸位点中,有插入/缺失35个,转换为34,颠换为21,转换/颠换比率为1.67。与NCBI上其它一些龟鳖的序列进行比对后,得到414bp的一致序列,其中236个为变异位点,总变异率为57.00%;简约信息位点181个。A、T、G、C的平均含量分别为35.7%、22.2%、18.1%、24.0%。A+T含量(57.9%)高于G+C含量(42.1%)。在414个核苷酸位点中,转换为30,颠换为14,转换/颠换比率为2.12。基于Kimura双参数模型(Kimura-2-parameter,K-2-P)分析龟鳖类种间、属间、科间遗传距离,并用邻接法构建分子系统树。结果显示:7种闭壳龟种间差异在0～0.043,平均为0.022;淡水龟科属间遗传距离为0.007～0.140,平均为0.074;曲颈龟亚目9个科间(不包括平胸龟)遗传距离为0.055～0.197,平均为0.139。由此认为,淡水龟科与陆龟科间亲缘关系比龟科更近;不支持将闭壳龟属拆分为闭壳龟属和盒龟属;支持将平胸龟属归为鳄龟科的一个属。2)16SrRNA:通过PCR扩增获得13种龟鳖类的线粒体16S rRNA基因片段序列,结合NCBI 47种龟鳖的同源序列一并分析。结果显示:60条序列比对排序后得到480bp的一致序列,其中有可变位点229个,总变异率为47.7%;简约信息位点186个,插入/缺失80个。4种碱基平均含量分别是:A-33.6%、T-24.3%、G-18.5%、C-23.7%,转换/颠换比率为1.95。7种闭壳龟种间的遗传距离为0.004～0.063,平均为0.03;潮龟科17个属间遗传距离为0.053～0.120,平均为0.091;曲颈龟亚目8个科间(不包括平胸龟)遗传距离为0.071～0.259,平均为0.169。用60条序列构建的邻接法(Neighbor-joining,NJ)、最大简约法(Maximum-parsimony,MP)和最大似然法(Maximum Likelihood,ML)系统树两两之间的拓扑结构均存在差异。本研究结果支持陆龟科与潮龟科组成一个单系类群,然后在与龟科构成姐妹群;支持将乌龟重新归入拟水龟属、将锯缘龟纳入闭壳龟属的观点;支持闭壳龟属不应拆分的观点,即不将黄缘盒龟和黄额盒龟归入盒龟属。这些均与依据12S rRNA序列所得结论相一致。然而本文支持将平胸龟属提升为单独的一个科,这与12S rRNA序列的结果不同。3)核基因:通过PCR扩增和测序的方法,获得2种龟的R35内含子部分序列,以及6种龟的Rag2基因部分序列。并结合NCBI其它龟鳖的同源序列一并分析。结果显示:R35序列比对后得到941bp的一致序列,Rag2比对后得到620bp的一致序列,二者合并、比对后得到1561bp的一致序列,其中共有505个可变核苷酸位点,总变异率为32.35%;简约信息位点239个,插入/缺失139个,转换/颠换比率为1.64。碱基A、T、G、C的平均含量分别为29.5%、28.5%、22.8%、19.2%。其中海龟科和鳄龟科之间K-2-P的遗传距离最小,为0.025,鳖科和南美侧颈龟科之间的遗传距离最大,为0.182。系统树显示,陆龟科与潮龟科构成一个单系类群,再与龟科形成姐妹群;支持陆龟总科与鳄龟科+海龟总科构成姐妹群,鳄龟科和海龟总科是姐妹群的关系。更多还原

【Abstract】 Mitochondrial 12SrRNA, 16SrRNA, and two nuclear genes (Rag2 and R35) sequence were utilized to analyze the molecular systematic characters of turtles.1) 12SrRNA: Mitochondrial 12SrRNA gene segment sequences of 15 Tortoises individuals were obtained by using PCR amplification and sequencing. After alignment, 433 bp consensus sequences of 12SrRNA were obtained. Among them, 191 variable sites were detected, accounting for 43.2% of total sequence, and 108 were parsim-informative sites. The average contents of A, T, G, and C were 21.46%, 34.42%, 25.85%, and 18.27%, respectively. The content of A+T(55.9%) is bigger than G+C(44.2%). In the 433 nucleotide sites, 34 transitional and 21 transversional sites with 35 indels were found, and the ratio between transition and transversion was 1.67. We downloaded the 12SrRNA sequences of other Tortoise species from NCBI and analyzed the phylogenetic relationships of them together with our own sequences. After alignment, 414 bp consensus sequences of 12SrRNA and 24.0%, respectively. The content of A+T (57.9%) is bigger than G+C (42.1%). 30 transition and 14 transversion in 414 nucleotide sites, and the ratio between transition and transversion were 2.12. Interspecific, intergenus, and interfamily genetic distances were calculated using the Kimura 2-parameter model with the pairwise deletion option. The NJ method was used to perform phylogenetic analysis. The results indicate that Bataguridae is closer to Testudinidae than that of Emydidae to Testudinidae. The results in this paper do not support the idea that the genus Cuora should be divided into two genera, Cuora and Cistoclemmys, and supporting the opinion that Platysternon should be refereed as a genus within Chelydridae.2) 16SrRNA: Mitochondrial DNA 16SrRNA gene segment sequences of 13 turtle individuals were obtained by using PCR amplification and sequencing. We downloaded the 16SrRNA sequences of other 47 turtles from NCBI and analyzed the phylogenetic characters of them together with our own 13 sequences. The genetic information indexes, including base composition,The ratio between transition and transversion (Ts/Tv ratio) and Kimura-2 parameter genetic distance (K-2-P),were calculated. 480bp consensus sequences of 16SrRNA were obtained from these 60 sequences after aligned. 229 variable sites were detected in all turtle individuals, accounting for 47.7% of total sites, with 186 parsim-informative and 80 indels. The average contents of A, T, G, and C were 33.6%, 24.3%, 18.5%, and 23.7%, respectively. The Ts/Tv ratio was 1.95. The K-2-P distances between the seven hinged turtles were 0.004～0.063, with average value 0.03; between 21 genus within Bataguridae were 0.035～0.120, with average 0.088; and between 8 families within Cryptodira (excluding Platysternon megacephalum) were 0.071～0.259, with average 0.169. Topologies of the NJ, MP and ML trees were different. The results indicate that 1) Family Emydidae is the sister taxon to Bataguridae plus Testudinidae, and Bataguridae is closer to Testudinidae than that of Emydidae to Testudinidae, 2) the taxonomica status of Chinemys should be revised, and classified into Genus Mauremys, and that Pyxidea should be classified into Cuora, 3) the results do not support the idea that the genus Cuora should be divided into two genera, i.e., Cuora and Cistoclemmys. Above conclusions are consistent with the results of mtDNA 12SrRNA sequences. However, this study supported the opinion that Platysternon should be elevated to family status (i.e. Platysternidae), contradicting with the study of mtDNA 12SrRNA sequences.3) Nuclear genes: R35 intron segment sequences of 2 turtles and Rag2 gene segment sequences of 6 turtle individuals were obtained by using PCR amplification and sequencing. We downloaded the homologous sequences of other turtles from NCBI and analyzed the phylogenetic characters of them together with our own sequences. The results are as follows: 941bp consensus sequences of R35 were obtained from these sequences after aligned. 620bp consensus sequences of Rag2 were obtained from these sequences after aligned. After aligned, 1561bp consensus sequences of combined R35/Rag2 date set ,505 variable sites were detected in all turtles individuals, accounting for 32.35%of total sites, with 239 parsim-informative and 139 indels. The average contents of A, T, G, and C were 29.5%,28.5%,22.8%and 19.2%, respectively. The Ts/Tv ratio was 1.64. The K-2-P distances between the Cheloniidae and Chelydridae was minimum (0.025); and The K-2-P distances between the Trionychidae and Podocnemididae was maximum (0.182). The phylogenetic trees indicate that 1) Family Emydidae is the sister taxon to Geoemydidae plus Testudinidae, 3) the results strong support that Testudinoidea is the sister taxon to Chelydridae plus Chelonioidea,2) the results also support that Family Chelydridae is the sister taxon to Chelonioidea.更多还原