【作者】 赵同领；

【导师】 黎学铭；

【作者基本信息】 广西医科大学 ， 病原生物学， 2007， 硕士

【摘要】 目的:研究广西扇棘单睾吸虫和其它人体吸虫5.8S rRNA序列和二级结构,探讨人体吸虫二级结构的特征,揭示单睾吸虫之间的亲缘关系。方法:采集广西扶绥县扇棘单睾吸虫,提取其DNA,PCR扩增5.8S rDNA序列并测序。另外选用Genbank登录的7种人体吸虫5.8S rDNA序列进行研究,包括:扇棘单睾吸虫、钩棘单睾吸虫、巨片形吸虫、裂体吸虫、獾似颈吸虫、卷棘口吸虫和猫后睾吸虫。应用DNAman软件把上述人体吸虫5.8SrDNA基因转换成5.8S rRNA。应用RNAstructure3.2软件,根据最小自由能原理,采用Zuker算法,构建5.8S rRNA分子二级结构。应用SPSSv14.0软件对5.8S rRNA一级结构和二级结构组成进行统计分析,人工比对分析部分人体吸虫5.8S rRNA序列和二级结构特征。结果:对广西扇棘单睾吸虫与Dzikowski等报道的扇棘单睾吸虫5.8SrRNA序列进行比较,显示第81、83、84、129、143位碱基发生变异,但这些突变碱基均位于5.8S rRNA二级结构未配对碱基区,碱基突变并未影响5.8S rRNA二级结构。对广西扇棘单睾吸虫与钩棘单睾吸虫5.8S rRNA序列进行比较,发现既存在未配对碱基区的变异又存在配对碱基区的变异,配对区碱基的变异使两者5.8S rRNA二级结构呈现较大差异。对复殖目部分吸虫5.8S rRNA二级结构进行比对分析,结果显示:5.8S rRNA二级结构具有相同的基本结构模式;Ⅰ分支具有1个保守模序为5′-GUGUCGAUG:CAUCGACAU-3′(钩棘单睾吸虫为5′-GUGUCGAUG:CAUCGAUAU-3′)。Ⅱ分支具有1个保守发卡结构,该发卡结构的茎是由9个碱基对组成的保守模序,S.sp保守模序为5′-GGCUACGGG:CCUGUGGCC-3′,其它7种保守模序为5′-GGCCAUGGG:CCUGUGGCC-3′;发卡环为富含尿嘧啶的6元环。在8种人体吸虫中,除裂体吸虫和猫后睾吸虫外,其它6种吸虫5.8SrRNA 5′端均有1段单链序列。在8种人体吸虫中,除钩棘单睾吸虫外,其余吸虫Ⅰ分支均有保守模序5′-GCAG:CUGC-3′。钩棘单睾吸虫和裂体吸虫与其它6种吸虫具有不同的H部和多分支环。巨片形吸虫、獾似颈吸虫和卷棘口吸虫Ⅰ分支尾端具有一个保守发卡结构。碱基的替代、丢失和插入多发生于5.8S rRNA二级结构单链区,双链区的替代、丢失和插入则往往会引起二级结构的较大变异。结论:本研究首次获得广西扇棘单睾吸虫5.8S rRNA基因序列,发现人体吸虫5.8S rRNA二级结构的一些特征。根据单睾吸虫二级结构特征,本研究还发现广西扇棘单睾吸虫与Dzikowski等报道的扇棘单睾吸虫亲缘关系比较近,与钩棘单睾吸虫亲缘关系比较远。更多还原

【Abstract】 OBJECTIVE:The The sequence and secondary structure of Haplorchis taichui from Guangxi and other Digenea Trematodes 5.8S rRNA are investigated to explore the kinships of Haplorchis trematodes and the secondary structure characters of Digenea Trematodes 5.8S rRNA.METHODS:Haplorchis taichuis were collected from Fusui county in Guangxi.The genomic DNA of Haplorchis taichui from Guangxi was extracted. The 5.8S rDNA was amplified by PCR and the sequences of PCR products were obtained by the sequencing reaction The 5.8S rDNAs of Haplorchis taichui,Haplorchis pumilio,Fasciola gigantica,Schistosoma sp,Isthmiophora melis,Echinostoma revolutum,Opisthorchis felineus from Genebank were investigated too.The 5.8S rDNAs of the above Trematodes are all transformed into the 5.8S rRNAs.The secondary structure of the 5.8S rRNAs were constructed using RNAstructure3.2 software according to the theory of minimally free energy with Zuker method.The composition of the sequence structures and the secondary structures of the 5.8S rRNAs were analyzed with SPSS v14.The sequences and secondary structures of Digenea Trematodes 5.8S rRNA were compared to explore their characters.RESULTS:Sequence comparing was performed between the 5.8S rRNA of Haplorchis taichui from Guangxi and that previously studied by Dzikowski,with the results showing some point mutations sites in the 81th, 83nd,84th,129th and 143th nucleotide.Those sites locate in unpaired regions so that they don’t affect on the secondary structural characters of the 5.8S rRNA. Sequence comparing between Haplorchis taichui from Guangxi and Haplorchis pumilio showed mutation sites which not only locate in unpaired regions but also in pared regions.The mutationof the nucleotides locating in pared regions cause more differences in 5.8S rRNA secondary structures between Haplorchis taichui from Guangxi and Haplorchis pumilio.The secondary structures of 5.8S rRNA in several Digenea Trematodes are all composed of main structure models. BranchⅠhas one conservative motif,5’-GUGUCGAUG:CAUCGACAU-3’ (5’-GUGUCGAUG:CAUCGAUAU-3’ in Haplorchis pumilio).BranchⅡhas a conservative hairpin,which consists of nine basepairs5’-GGCCAUGGG: CCUGUGGCC-3’(5’-GGCUACGGG:CCUGUGGCC-3’ in Schistosoma sp) The hairpin loop is a six-membered ring of U rich.There is a single strand sequence in the 5’ end of 5.8S rRNA among the above Trematodes except Schistosoma sp and Opisthorchis felineus.The Trematodes except Haplorchis taichui have a conservative motif(5’-GCAG:CUGC-3’)in branchⅠ. Haplorchis taichui and Schistosoma sp have a different handle and multibranch loop,compared with other six Trematodes.The branchⅠend of Fasciola gigantica,Isthmiophora melis and Echinostoma revolutum have a conservative hairpin.Replacement,deletion and insertion of bases mostly occur in the single strand region of the secondary structure of the 5.8S rRNAs.While these occurre in double strand,the secondary structure changes tremendously.CONCLUSION:It was the first time that this study had obtainded 5.8S rRNA gene sequence of Haplorchis taichui from Guangxi.The secondary structure characters of 5.8S rRNA were discovered in Digenea Trematodes. According to their secondary structure characters of 5.8S rRNA,the kinship between Haplorchis taichui from Guangxi and that previously studied by Dzikowski is nearer while the kinship between Haplorchis taichui from Guangxi and Haplorchis pumilio is more far away.更多还原