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林业科学 ›› 2015, Vol. 51 ›› Issue (5): 127-134.doi: 10.11707/j.1001-7488.20150515

• 研究简报 • 上一篇    下一篇

毛竹微型颠倒重复序列转座子PhTourist1的克隆与分析

胡慧, 周明兵, 杨萍, 汤定钦   

  1. 浙江农林大学 亚热带森林培育国家重点实验室培育基地 临安 311300
  • 收稿日期:2014-09-01 修回日期:2015-01-19 出版日期:2015-05-25 发布日期:2015-06-11
  • 通讯作者: 周明兵
  • 基金资助:

    国家自然科学基金项目"毛竹活性MITE的分离及与宿主基因表达调控网络互作机制解析"(31270645);国家自然科学基金项目"毛竹LTR反转录转座子转座调控机理及对宿主生物多样性的影响"(31470615);浙江自然科学基金杰出青年项目"竹亚科LTR反转录座子的分布和增殖模式及对宿主基因组进化影响"(LR12C16001);浙江自然科学基金项目"Mariner-like转座酶的人工定向进化"(Y3100239);浙江省重大科技专项"雷竹等中小径散生笋用竹新品种选育及推广"(2012C12908-2)。

Cloning and Analysis of Miniature Inverted Repeat Transposable Elements PhTourist 1 from Phyllostachys edulis

Hu Hui, Zhou Mingbing, Yang Ping, Tang Dingqin   

  1. The Nurturing Station for the State Key Laboratory of Subtropical Silviculture Zhejiang A & F University Lin'an 311300
  • Received:2014-09-01 Revised:2015-01-19 Online:2015-05-25 Published:2015-06-11

摘要:

【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——PhTourist 1为例,研究PhTourist 1的结构特点和转座特性,并分析PhTourist 1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PhTourist 1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PhTourist 1在毛竹基因组中的插入时间和分布情况。通过PCR手段确认PhTourist 1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PhTourist 1-3,通过RTFQ PCR(real-time fluorescence quantitative PCR)分析PhTourist 1-3存在与否对其下游基因表达量的影响。【结果】 共得到30个PhTourist 1,通过生物信息学技术与手段确定得到的PhTourist 1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PhTourist 1的插入时间与分布分析了解到PhTourist 1在毛竹基因组中的插入时间相对集中,且PhTourist 1偏好插在基因附近。对毛竹基因组中30个PhTourist 1的验证确认了每个位置的正确性,并发现PhTourist 1-3存在插入多态性。且PhToutist 1-3的切除为完整切除,与mPing的特性一致。利用RTFQ PCR对PhTourist 1-3下游基因PH01000402G0860表达量的分析结果表明PhTourist 1-3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PhTourist 1,其中PhTourist 1-3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PhTourist 1-3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。

关键词: 毛竹, Tourist-MITE, mPing, 转座, 基因表达

Abstract:

【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 PhTourist 1 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 PhTourist 1 were identified and the insert preference of PhTourist 1 was investigated using software of MEGA5.1, DNAStar and WebLogo 3-Create. Then the insertion time and distribution of PhTourist 1 in the P. edulis genome were calculated. PhTourist 1 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 PhTourist 1-3 was analyzed with PhTourist 1-3 exists or not.【Result】 30 copies of PhTourist 1 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 PhTourist 1 in P. edulis genome was relatively concentrated. PhTourist 1 also preferentially inserts near genes. Each of the insertion sites of PhTourist 1 was verified correct, while only PhTourist 1-3 has insert polymorphism among the 30 copies. There was no footprint at the excision site of PhTourist 1-3 which was in cosistence with mPing. The analysis by RTFQ PCR of the expression level of a downstream gene PH01000402G0860 of PhTourist 1-3 revealed an increase by 8.04 times with the missing of PhTourist 1-3 than the existing of PhTourist 1-3. 【Conclusion】 PhTourist 1-3, one of the identified PhTourist 1 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.

Key words: Phyllostachys edulis, Tourist-MITE, mPing, transposition, gene expression

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