论文编号:SP016论文字数:9299,页数:21
摘 要：本试验以文蛤（M.meretrix）闭壳肌为材料，采用CTAB裂解法提取基因组总DNA，根据COI基因的保守序列设计引物，PCR扩增文蛤的COI基因片段序列。PCR产物送上海生工进行纯化回收，并采用扩增引物进行正反双向测序。用DNAStar （version 5.01）中的SeqMan软件对正反序列进行组装，得到COI基因片段的一致序列，长度为709bp（包括引物在内）。用EditSeq对所得到的序列进行编辑和分析，序列中A=21.44%， G=20.17%，T=44.85%，C=13.54%，A+T=66.29%，G+C=33.71%，使用无脊椎动物线粒体遗传密码把COI基因片段翻译成氨基酸序列，此片段共编码235个氨基酸。进入GenBank对得到的序列进行BLAST，以确定所得到的序列是COⅠ基因序列，并与亲缘关系在74%以上的帘蛤科的12个不同种COⅠ基因序列用MegAlign软件进行比对，并以住石蛤科的住石蛤相应序列数据为外群构建进化树。系统发育树显示：文蛤和同属的斧文蛤和丽文蛤聚在一起，这和传统分类吻合。
关键词： 文蛤 COI基因 序列分析
Sequence Analysis of Cytochrome Oxidase Subunit I
Gene Fragment of Meretrix meretrix
Abstract: The whole DNA of M. meretrix was extracted using CTAB method from adductor muscle. And the COI gene fragment were amplified by PCR and sequenced in Shanghai Sangon. The result showed that size of COI gene fragment was 709bp (contain the primer), the A, T, G and C contents in this fragment were 21.44%, 44.85%, 20.17% and 13.54% respectively. The A+T content was higher than G+C content. The same sequences of 12 Veneridae clams were downloaded from GenBank which have above 74% homology with M. meretrix.The homologus sequences were aligned, and the molecular phylogenetic tree was constructed with the sequence information of petricola lapicida as outgroup using MegAlign software. Tree topologies indicated that M. meretrix shared a close relationship with Meretrix lusoria and Meretrix lamarckii. It is consistent with the morphological and taxonomic results.
Keywords: M. meretrix; Cytochrome Oxidase Subunit I Gene; Sequence analysis.
目 录
1 引言……………………………………………………………………………………… 1
文蛤的生物学特性………………………………………………………………………… 1
文蛤的药用价值………………………………………………………………… 2
本试验的目的意义…………………………………………………………………… 2
2 材料和方法…………………………………………………………………………… 3
2.1 材料 ………………………………………………………………………………… 3
2.2方法…………………………………………………………………………………… 3
3 结果和分析……………………………………………………………………………… 5
3.1文蛤基因组总DNA电泳图……………………………………………………………… 5
3.2 文蛤PCR电泳结果………………………………………………………………………… 6
3.3测序及序列组装…………………………………………………………………………… 6
3.4 序列比对…………………………………………………………………………………… 7
3.5构建系统发育进化树…………………………………………………………………… 10
4 讨论 ………………………………………………………………………………… 11
4.1 DNA提取……………………………………………………………………………… 11
4.2 PCR中遇到的问题……………………………………………………………………… 12
4.3 COI基因序列的应用…………………………………………………………………… 12
结论 …………………………………………………………………………………… 14
致谢 …………………………………………………………………………………… 15
参考文献……………………………………………………………………………… 16
文蛤COI基因片段序列分析......