CAJ | 학술논문

【目的】微型颠倒重复序列( MITE)是 DNA转座子中一类依赖自主转座子提供的转座酶进行转座的非自主转座子。以毛竹中与水稻 mPing同源的 MITE———PhTourist1为例，研究 PhTourist1的结构特点和转座特性，并分析 PhTourist1的转座对其附近基因表达的影响，进而了解转座子对毛竹基因组多态性形成和基因表达调控的作用。【方法】利用 MEGA5.1，DNAStar，WebLogo 3-Create等软件对 PhTourist1的末端反向重复序列(TIR)、靶位点重复序列( TSD)、插入位点序列偏好性等进行系统的研究和分析，并计算 PhTourist1在毛竹基因组中的插入时间和分布情况。通过 PCR手段确认 PhTourist1在来自同一母本的24株毛竹实生苗基因组中的所有位置并验证是否存在插入多态性。针对其中发生转座的 PhTourist1-3，通过 RTFQ PCR ( real-time fluorescence quantitative PCR)分析PhTourist1-3存在与否对其下游基因表达量的影响。【结果】共得到30个 PhTourist1，通过生物信息学技术与手段确定得到的 PhTourist1的 TIR(GGCCAGTCTCAATG)、TSD(TWA)及插入偏好性序列((C/T)T(C/A)T(T/A)A(G/T) A ( A/C ))与已报道的 Tourist-like MITE 的特点吻合。通过对 PhTourist1的插入时间与分布分析了解到PhTourist1在毛竹基因组中的插入时间相对集中，且 PhTourist1偏好插在基因附近。对毛竹基因组中30个PhTourist1的验证确认了每个位置的正确性，并发现 PhTourist1-3存在插入多态性。且 PhToutist1-3的切除为完整切除，与 mPing的特性一致。利用 RTFQ PCR对 PhTourist1-3下游基因 PH01000402G0860表达量的分析结果表明PhTourist1-3转座后 PH01000402G0860的表达量比其转座前增加了8.04倍。【结论】本研究鉴定到的与 mPing 高度同源的转座子 PhTourist1，其中 PhTourist1-3在毛竹基因组中可以发生转座，是首次在毛竹基因组中发现的有活性的 Tourist-like MITE，且 PhTourist1-3的转座对宿主基因表达产生显著的影响。这些结果表明，作为毛竹基因组重要组成部分的转座子可以通过自身转座参与宿主基因组多态性的形成，调节宿主基因表达，进而参与毛竹生长发育的调控。【目的】微型顛倒重複序列( MITE)是 DNA轉座子中一類依賴自主轉座子提供的轉座酶進行轉座的非自主轉座子。以毛竹中與水稻 mPing同源的 MITE———PhTourist1為例，研究 PhTourist1的結構特點和轉座特性，併分析 PhTourist1的轉座對其附近基因錶達的影響，進而瞭解轉座子對毛竹基因組多態性形成和基因錶達調控的作用。【方法】利用 MEGA5.1，DNAStar，WebLogo 3-Create等軟件對 PhTourist1的末耑反嚮重複序列(TIR)、靶位點重複序列( TSD)、插入位點序列偏好性等進行繫統的研究和分析，併計算 PhTourist1在毛竹基因組中的插入時間和分佈情況。通過 PCR手段確認 PhTourist1在來自同一母本的24株毛竹實生苗基因組中的所有位置併驗證是否存在插入多態性。針對其中髮生轉座的 PhTourist1-3，通過 RTFQ PCR ( real-time fluorescence quantitative PCR)分析PhTourist1-3存在與否對其下遊基因錶達量的影響。【結果】共得到30箇 PhTourist1，通過生物信息學技術與手段確定得到的 PhTourist1的 TIR(GGCCAGTCTCAATG)、TSD(TWA)及插入偏好性序列((C/T)T(C/A)T(T/A)A(G/T) A ( A/C ))與已報道的 Tourist-like MITE 的特點吻閤。通過對 PhTourist1的插入時間與分佈分析瞭解到PhTourist1在毛竹基因組中的插入時間相對集中，且 PhTourist1偏好插在基因附近。對毛竹基因組中30箇PhTourist1的驗證確認瞭每箇位置的正確性，併髮現 PhTourist1-3存在插入多態性。且 PhToutist1-3的切除為完整切除，與 mPing的特性一緻。利用 RTFQ PCR對 PhTourist1-3下遊基因 PH01000402G0860錶達量的分析結果錶明PhTourist1-3轉座後 PH01000402G0860的錶達量比其轉座前增加瞭8.04倍。【結論】本研究鑒定到的與 mPing 高度同源的轉座子 PhTourist1，其中 PhTourist1-3在毛竹基因組中可以髮生轉座，是首次在毛竹基因組中髮現的有活性的 Tourist-like MITE，且 PhTourist1-3的轉座對宿主基因錶達產生顯著的影響。這些結果錶明，作為毛竹基因組重要組成部分的轉座子可以通過自身轉座參與宿主基因組多態性的形成，調節宿主基因錶達，進而參與毛竹生長髮育的調控。
【목적】미형전도중복서렬( MITE)시 DNA전좌자중일류의뢰자주전좌자제공적전좌매진행전좌적비자주전좌자。이모죽중여수도 mPing동원적 MITE———PhTourist1위례，연구 PhTourist1적결구특점화전좌특성，병분석 PhTourist1적전좌대기부근기인표체적영향，진이료해전좌자대모죽기인조다태성형성화기인표체조공적작용。【방법】이용 MEGA5.1，DNAStar，WebLogo 3-Create등연건대 PhTourist1적말단반향중복서렬(TIR)、파위점중복서렬( TSD)、삽입위점서렬편호성등진행계통적연구화분석，병계산 PhTourist1재모죽기인조중적삽입시간화분포정황。통과 PCR수단학인 PhTourist1재래자동일모본적24주모죽실생묘기인조중적소유위치병험증시부존재삽입다태성。침대기중발생전좌적 PhTourist1-3，통과 RTFQ PCR ( real-time fluorescence quantitative PCR)분석PhTourist1-3존재여부대기하유기인표체량적영향。【결과】공득도30개 PhTourist1，통과생물신식학기술여수단학정득도적 PhTourist1적 TIR(GGCCAGTCTCAATG)、TSD(TWA)급삽입편호성서렬((C/T)T(C/A)T(T/A)A(G/T) A ( A/C ))여이보도적 Tourist-like MITE 적특점문합。통과대 PhTourist1적삽입시간여분포분석료해도PhTourist1재모죽기인조중적삽입시간상대집중，차 PhTourist1편호삽재기인부근。대모죽기인조중30개PhTourist1적험증학인료매개위치적정학성，병발현 PhTourist1-3존재삽입다태성。차 PhToutist1-3적절제위완정절제，여 mPing적특성일치。이용 RTFQ PCR대 PhTourist1-3하유기인 PH01000402G0860표체량적분석결과표명PhTourist1-3전좌후 PH01000402G0860적표체량비기전좌전증가료8.04배。【결론】본연구감정도적여 mPing 고도동원적전좌자 PhTourist1，기중 PhTourist1-3재모죽기인조중가이발생전좌，시수차재모죽기인조중발현적유활성적 Tourist-like MITE，차 PhTourist1-3적전좌대숙주기인표체산생현저적영향。저사결과표명，작위모죽기인조중요조성부분적전좌자가이통과자신전좌삼여숙주기인조다태성적형성，조절숙주기인표체，진이삼여모죽생장발육적조공。
Objective]Miniature inverted-repeat transposable elements ( MITEs) ,some of which are known as active non-autonomous DNA transposons,can jump by transposases encoded by the autonomous DNA transposons. It has been reported that the transposition of MITE influences the expression of host ’s genes. In order to study the structural characteristics and transposition characteristics of MITE,and to analyze the impact of the transposition of MITE on expression of its near genes in Phyllostachys edulis genome,the Tourist-like MITE PhTourist1 which was homologous with mPing from rice was chosen to analyze the role of transposons in genome polymorphism formation and gene expression regulation in P. edulis. [Method]TIR ( terminal inverted repeat) and TSD ( target site duplication) of PhTourist1 were identified and the insert preference of PhTourist1 was investigated using software of MEGA5. 1,DNAStar and WebLogo 3-Create. Then the insertion time and distribution of PhTourist1 in the P. edulis genome were calculated. PhTourist1 insertion sites in the P. edulis genome were tested by PCR in the 24 seedlings to test their insert polymorphisms. With the real-time fluorescence quantitative PCR ( RTFQ PCR) technique,the expression level of gene downstream PhTourist1-3 was analyzed with PhTourist1-3 exists or not. [Result]30 copies of PhTourist1 were discovered. Their TIR ( GGCCAGTCTCAATG) ,TSD ( TWA ) and insert preference sequence ( ( C/T ) T ( C/A ) T ( T/A ) A ( G/T ) A ( A/C ) ) identified with bioinformatics tools were consistent with previous reports. The insert time of PhTourist1 in P. edulis genome was relatively concentrated. PhTourist1 also preferentially inserts near genes. Each of the insertion sites of PhTourist1 was verified correct,while only PhTourist1-3 has insert polymorphism among the 30 copies. There was no footprint at the excision site of PhTourist1-3 which was in cosistence with mPing. The analysis by RTFQ PCR of the expression level of a downstream gene PH01000402 G0860 of PhTourist1-3 revealed an increase by 8. 04 times with the missing of PhTourist1-3 than the existing of PhTourist1-3 . [Conclusion]PhTourist1-3 ,one of the identified PhTourist1 homology with mPing in the study,could jump in the genome of P. edulis. It was the first active Tourist-like MITE discovered in P. edulis and had significant influence on expression of its downstream genes. The results showed that transposons as an important part of P. edulis genome could participate in the formation of gene polymorphism,regulate the gene expression and involve in the regulatory of host growth and development.