毛竹微型颠倒重复序列转座子PhTourist1的克隆与分析

Cloning and Analysis of Miniature Inverted Repeat Transposable Elements Ph Tourist1 from Phyllostachys edulis

【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m Ping同源的MITE——Ph Tourist1为例,研究Ph Tourist1的结构特点和转座特性,并分析Ph Tourist1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,Web Logo 3-Create等软件对Ph Tourist1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Ph Tourist1在毛竹基因组中的插入时间和分布情况。通过PCR手段确认Ph Tourist1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Ph Tourist1-3,通过RTFQ PCR(real-time fluorescence quantitative PCR)分析Ph Tourist1-3存在与否对其下游基因表达量的影响。【结果】共得到30个Ph Tourist1,通过生物信息学技术与手段确定得到的Ph Tourist1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Ph Tourist1的插入时间与分布分析了解到Ph Tourist1在毛竹基因组中的插入时间相对集中,且Ph Tourist1偏好插在基因附近。对毛竹基因组中30个Ph Tourist1的验证确认了每个位置的正确性,并发现Ph Tourist1-3存在插入多态性。且Ph Toutist1-3的切除为完整切除,与m Ping的特性一致。利用RTFQ PCR对Ph Tourist1-3下游基因PH01000402G0860表达量的分析结果表明Ph Tourist1-3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m Ping高度同源的转座子Ph Tourist1,其中Ph Tourist1-3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Ph Tourist1-3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。

[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.