热带医学杂志
熱帶醫學雜誌
열대의학잡지
JOURNAL OF TROPICAL MEDICINE
2008年
7期
633-638,648
,共7页
罗丽莉%徐晓园%许铭炎%傅玉才
囉麗莉%徐曉園%許銘炎%傅玉纔
라려리%서효완%허명염%부옥재
阴道毛滴虫%Rab1-like基因%25 bp内含子
陰道毛滴蟲%Rab1-like基因%25 bp內含子
음도모적충%Rab1-like기인%25 bp내함자
Trichomonas vaginalis%Rab1 gene%25 bp intron
目的 克隆和鉴定一个新的阴道毛滴虫Rab1-like基因(TvRab1-like)及其内含子.方法 我们从一阴道毛滴虫cDNA表达文库中分离出一个cDNA克隆,它与各物种的Rab家族蛋白有较高的同源性,因此我们进一步用BLASTP、RPS-BLAST、ClustalW和MEGA3等分析软件对该cDNA克隆进行了序列分析和进化树分析;用PCR和RT-PCR等技术分别对该基因组和mRNA进行了扩增和测序分析.结果 序列分析结果表明该cDNA克隆长705 bp,开放阅读框具603 bp,推测肽链含有200个氨基酸.序列比较分析结果提示该cDNA克隆所推测的蛋白质是一个Rab1亚家族的亚型.进化树分析也表明它属于阴道毛滴虫Rab1亚家族.基因组PCR扩增和测序分析表明该基因包含一个25 bp的内含子,该内含子具有阴道毛滴虫和其它真核生物较大内含子所具备的典型的5'GT-AG-3'和分支位点基序.RT-PCR产物及其测序分析表明在该基因的转录本中存在着未剪切和剪切后的mRNA,说明确实有内含子的存在.结论 TvRab1-like基因属于阴道毛滴虫Rab1亚家族,该基因含有一个25 bp的内含子.该内含子是至今发现的最小的阴道毛滴虫基因内含子之一,很可能也是真核生物中最小的内含子.对诸类最低等真核生物内含子的研究将有助于我们理解真核生物内含子的起源和进化.
目的 剋隆和鑒定一箇新的陰道毛滴蟲Rab1-like基因(TvRab1-like)及其內含子.方法 我們從一陰道毛滴蟲cDNA錶達文庫中分離齣一箇cDNA剋隆,它與各物種的Rab傢族蛋白有較高的同源性,因此我們進一步用BLASTP、RPS-BLAST、ClustalW和MEGA3等分析軟件對該cDNA剋隆進行瞭序列分析和進化樹分析;用PCR和RT-PCR等技術分彆對該基因組和mRNA進行瞭擴增和測序分析.結果 序列分析結果錶明該cDNA剋隆長705 bp,開放閱讀框具603 bp,推測肽鏈含有200箇氨基痠.序列比較分析結果提示該cDNA剋隆所推測的蛋白質是一箇Rab1亞傢族的亞型.進化樹分析也錶明它屬于陰道毛滴蟲Rab1亞傢族.基因組PCR擴增和測序分析錶明該基因包含一箇25 bp的內含子,該內含子具有陰道毛滴蟲和其它真覈生物較大內含子所具備的典型的5'GT-AG-3'和分支位點基序.RT-PCR產物及其測序分析錶明在該基因的轉錄本中存在著未剪切和剪切後的mRNA,說明確實有內含子的存在.結論 TvRab1-like基因屬于陰道毛滴蟲Rab1亞傢族,該基因含有一箇25 bp的內含子.該內含子是至今髮現的最小的陰道毛滴蟲基因內含子之一,很可能也是真覈生物中最小的內含子.對諸類最低等真覈生物內含子的研究將有助于我們理解真覈生物內含子的起源和進化.
목적 극륭화감정일개신적음도모적충Rab1-like기인(TvRab1-like)급기내함자.방법 아문종일음도모적충cDNA표체문고중분리출일개cDNA극륭,타여각물충적Rab가족단백유교고적동원성,인차아문진일보용BLASTP、RPS-BLAST、ClustalW화MEGA3등분석연건대해cDNA극륭진행료서렬분석화진화수분석;용PCR화RT-PCR등기술분별대해기인조화mRNA진행료확증화측서분석.결과 서렬분석결과표명해cDNA극륭장705 bp,개방열독광구603 bp,추측태련함유200개안기산.서렬비교분석결과제시해cDNA극륭소추측적단백질시일개Rab1아가족적아형.진화수분석야표명타속우음도모적충Rab1아가족.기인조PCR확증화측서분석표명해기인포함일개25 bp적내함자,해내함자구유음도모적충화기타진핵생물교대내함자소구비적전형적5'GT-AG-3'화분지위점기서.RT-PCR산물급기측서분석표명재해기인적전록본중존재착미전절화전절후적mRNA,설명학실유내함자적존재.결론 TvRab1-like기인속우음도모적충Rab1아가족,해기인함유일개25 bp적내함자.해내함자시지금발현적최소적음도모적충기인내함자지일,흔가능야시진핵생물중최소적내함자.대제류최저등진핵생물내함자적연구장유조우아문리해진핵생물내함자적기원화진화.
Objective The aim of this study is to clone and characterize a novel Trichomonas vaginalis Rabl-like geue (TvRabl-like) with a small intron. Methods The eDNA clone of TvRabl-like gene was isolated from a cDNA expression library and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST ClustalW programs.Phylogenetic analysis was carried out by MEGA3 program. The genomic DNA and mRNA of TvRab1-like gene were amplified using PCR and RT-PCR techniques respectively and also sequenced. Results The eDNA sequence of TvRab1-like gene had a length of 705 base pairs with an open reading frame of 603 bp. The deduced amino acid sequence from the open reading frame possessed 200 residuals corresponding to a putative M.W. 22532.2 and an estimated pl of 7.4. Sequence analysis demonstrated that TvRab1-like gene showed the highest homology to T. vaginalis Rabla (63% identity and 79% similarity) and the Rabl subfamily of other species, suggesting that the deduced amino acid sequence from this cDNA clone was a Rabl isoform. Phylogenetic analysis showed that TvRab1-like gene was clustered with T.vaginalis Rab1 subfamily in the phylogenetic tree. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA possessed a 25 bp intron which contained canonical 5' GT-AG-3' and branch site motifs as those larger introns found in T.vaginalis and other eukaryotes. The analysis of RT-PCR products demonstrated the presence of the unspliced mRNA and spliced mRNA, indicating that there was a intron. Conclusion These data suggest that TvRabl-like gene belongs to T.vaginalis Rabl subfamily. TvRabl-like gene possesses a 25 bp splieeosomal intron which is the smallest one of the introns identified in this deepest-branching protist and might be the shortest intron of eukaryotes. Study of those introns might provide more insights into the intron evolution of eukaryotes.
