菲律宾蛤仔18S rRNA基因PCR扩增及序列分析

范文编号:SP014  范文字数:8926,页数:23

摘  要：本试验采用CTAB裂解法提取了菲律宾蛤仔的总DNA。根据18S基因的保守序列设计3段引物，进行PCR扩增，并采用扩增引物进行正反双向测序。通过DNAstar Package中的SeqMan软件对序列进行组装,可以得到18S rRNA基因全序列。在美国国家信息中心（NCBI）网站上对得到的序列进行BLAST，以确定所得到的序列是18S基因序列。结果显示，菲律宾蛤仔的18S全序列长度为1833bp，A占23.95％，G占27.61％，C占24.22％，T占24.22％，A+T=48.17%,C+G=51.83%。最后用MegAlign软件对菲律宾蛤仔与一些相近的双壳纲软体动物的18S基因序列进行比对，序列对比显示菲律宾蛤仔与同一帘蛤科的疣帘蛤及薄片镜蛤的18S序列同源性较高，其次是青蛤和蚬科的河蚬。系统发育进化树显示帘蛤目与海螂目的亲缘关系较近，与珍珠贝目亲缘关系较远。但总的来说，它们的同源性都在85%以上，从而进一步证明了18S基因序列是具有高度相似性的保守序列。

关键词：菲律宾蛤仔  PCR  18S rRNA基因  序列分析　分子系统发育

The PCR amplify and sequences analysis of 18S rRNA gene of
Ruditapes philippinarum

Abstract: The genome DNA was extracted from the adductor muscle of Ruditapes philippinarum with the method of CTAB. Three primers was designed according to the conversation of 18S gene sequences, which was used in the PCR. Then the sequence was sequenced in both direction and by using SeqMan software in DNAstar Package ,the sequences was assembled to get the complete sequence of 18S ribosomal RNA gene. The 18S gene could be ascertain by carring out BLAST on the NCBI web site. The result showed that length of the 18S ribosomal RNA gene of Ruditapes philippinarum is 1833 bp , in which A account for 23.95% , G accounts for 27.61% , C accounts for 24.22% , T accounts for 24.22%, A + T = 48.17%, C +G = 51.83%. Compared with other available 18S gene of  Bivalvia species by MegAlign software, it was find out that the 18S ribosomal RNA gene of Ruditapes philippinarum has a high congenetic with Dosinia corrugate ,Venus verrucosa , Cyclina sinensis and Corbicula fluminea ,which were all in Veneroida. The phylogenetic tree showed that Veneroida has a closer relationship with Myoida than Pterioida. In conclusion, they all have a high homology above 85%, which further proofed that the 18S ribosomal RNA gene sequences is very conservative.

Keywords: Ruditapes philippinarum；PCR；18S rRNA genes；Sequences analysis；Molecular phylogeny．

  方正姚体目  录
    
1 引言………………………………………………………………………………………1
1.1  研究意义……………………………………………………………………………1
1.2  研究历史和现状……………………………………………………………………2
2 试验材料与方法…………………………………………………………………………3
2.1 试验材料………………………………………………………………………………3
2.2 试验器材………………………………………………………………………………3
2.3 总DNA提取…………………………………………………………………………3
2.4 DNA电泳检测……………………………………………………………………… 4
2.5 PCR扩增…………………………………………………………………………… 5
2.6 PCR产物检测……………………………………………………………………6
2.7测序……………………………………………………………………………………6
2.8序列分析………………………………………………………………………………6
3 试验结果及分析…………………………………………………………………………7
3.1 DNA提取结果……………………………………………………………………… 7
3.2 PCR结果………………………………………………………………………………7
3.3测序结果…………………………………………………………………………… 7
3.4序列分析结果………………………………………………………………………10
4 讨论…………………………………………………………………………………… 14
4.1 DNA提取…………………………………………………………………………… 14
4.2 PCR扩增中遇到的问题…………………………………………………………… 15
4.3存在问题与展望……………………………………………………………………15
结论………………………………………………………………………………………16
致谢……………………………………………………………………………………… 17
参考文献………………………………………………………………………………… 18

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