菲律賓蛤仔18S rRNA基因PCR擴增及序列分析

食品科學論文編號:SP014  論文字數:8926,頁數:23

  方正姚體目  錄
    
1 引言………………………………………………………………………………………1
1.1  研究意義……………………………………………………………………………1
1.2  研究歷史和現狀……………………………………………………………………2
2 試驗材料與方法…………………………………………………………………………3
2.1 試驗材料………………………………………………………………………………3
2.2 試驗器材………………………………………………………………………………3
2.3 總DNA提取…………………………………………………………………………3
2.4 DNA電泳檢測……………………………………………………………………… 4
2.5 PCR擴增…………………………………………………………………………… 5
2.6 PCR產物檢測……………………………………………………………………6
2.7測序……………………………………………………………………………………6
2.8序列分析………………………………………………………………………………6
3 試驗結果及分析…………………………………………………………………………7
3.1 DNA提取結果……………………………………………………………………… 7
3.2 PCR結果………………………………………………………………………………7
3.3測序結果…………………………………………………………………………… 7
3.4序列分析結果………………………………………………………………………10
4 討論…………………………………………………………………………………… 14
4.1 DNA提取…………………………………………………………………………… 14
4.2 PCR擴增中遇到的問題…………………………………………………………… 15
4.3存在問題與展望……………………………………………………………………15
結論………………………………………………………………………………………16
致謝……………………………………………………………………………………… 17
參考文獻………………………………………………………………………………… 18

摘  要：本試驗采用CTAB裂解法提取了菲律賓蛤仔的總DNA。根據18S基因的保守序列設計3段引物，進行PCR擴增，并采用擴增引物進行正反雙向測序。通過DNAstar Package中的SeqMan軟件對序列進行組裝,可以得到18S rRNA基因全序列。在美國國家信息中心（NCBI）網站上對得到的序列進行BLAST，以確定所得到的序列是18S基因序列。結果顯示，菲律賓蛤仔的18S全序列長度為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軟件對菲律賓蛤仔與一些相近的雙殼綱軟體動物的18S基因序列進行比對，序列對比顯示菲律賓蛤仔與同一簾蛤科的疣簾蛤及薄片鏡蛤的18S序列同源性較高，其次是青蛤和蜆科的河蜆。系統發育進化樹顯示簾蛤目與海螂目的親緣關系較近，與珍珠貝目親緣關系較遠。但總的來說，它們的同源性都在85%以上，從而進一步證明了18S基因序列是具有高度相似性的保守序列。

關鍵詞：菲律賓蛤仔  PCR  18S rRNA基因  序列分析　分子系統發育

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．