为探明miRNA在红麻中的功能,以红麻基因组DNA为模板,根据红麻microRNA(miRNA)高通量测序数据结果,克隆红麻miR394前体基因序列,并构建CRISPR/Cas9载体。结果表明红麻miR394前体基因序列大小为175bp,用Mfold在线软件分析,其前体序列能形...为探明miRNA在红麻中的功能,以红麻基因组DNA为模板,根据红麻microRNA(miRNA)高通量测序数据结果,克隆红麻miR394前体基因序列,并构建CRISPR/Cas9载体。结果表明红麻miR394前体基因序列大小为175bp,用Mfold在线软件分析,其前体序列能形成稳定的二级结构。根据红麻miR394前体基因序列,设计合适的CRISPR/Cas9靶标序列,利用改良的CRISPR/Cas9多靶点载体系统,以AtU3b和AtU6-29质粒为模板,使用Overlapping PCR法构建AtU3b-sgRNA和AtU6-29-sgRNA表达盒,使用Golden Gate Cloning方法成功把靶标AtU3b-sgRNA和AtU6-29-sgRNA表达盒构建到pYLCRISPR/Cas9载体中,并将构建好的载体命名为pCas9-miR394。分离红麻叶片原生质体,将pCas9-miR394载体转入红麻叶片原生质体中进行瞬时表达,测序结果表明:2个靶点Target site 1(T1)和Target site 2(T2)处都发生碱基突变,表明pCas9-miR394载体在红麻原生质体中具有靶向切割的活性。展开更多
Rice leaf inclination is an important agronomic trait, closely related to plant architecture and yield.Identification of genes controlling leaf inclination would assist in crop improvement. Although various factors,in...Rice leaf inclination is an important agronomic trait, closely related to plant architecture and yield.Identification of genes controlling leaf inclination would assist in crop improvement. Although various factors,including the plant hormones auxin and brassinosteroids,have been shown to regulate lamina joint development,the role of microRNAs in regulating leaf inclination remains largely unknown. Here, we functionally characterize the role of rice miR394 and its target, LEAF INCLINCATION 4(LC4), which encodes an F-box protein, in the regulation of leaf inclination. We show that miR394 and LC4 work,antagonistically, to regulate leaf lamina joint development and rice architecture, by modulating expansion and elongation of adaxial parenchyma cells. Suppressed expression of miR394, or enhanced expression of LC4,results in enlarged leaf angles, whereas reducing LC4 expression by CRISPR/Cas9 leads to reduced leaf inclination, suggesting LC4 as candidate for use in rice architecture improvement. LC4 interacts with SKP1, a component of the SCF E3 ubiquitin ligase complex, and transcription of both miR394 and LC4 are regulated by auxin. Rice plants with altered expression of miR394 or LC4 have altered auxin responses, indicating that the miR394-LC4 module mediates auxin effects important for determining rice leaf inclination and architecture.展开更多
文摘为探明miRNA在红麻中的功能,以红麻基因组DNA为模板,根据红麻microRNA(miRNA)高通量测序数据结果,克隆红麻miR394前体基因序列,并构建CRISPR/Cas9载体。结果表明红麻miR394前体基因序列大小为175bp,用Mfold在线软件分析,其前体序列能形成稳定的二级结构。根据红麻miR394前体基因序列,设计合适的CRISPR/Cas9靶标序列,利用改良的CRISPR/Cas9多靶点载体系统,以AtU3b和AtU6-29质粒为模板,使用Overlapping PCR法构建AtU3b-sgRNA和AtU6-29-sgRNA表达盒,使用Golden Gate Cloning方法成功把靶标AtU3b-sgRNA和AtU6-29-sgRNA表达盒构建到pYLCRISPR/Cas9载体中,并将构建好的载体命名为pCas9-miR394。分离红麻叶片原生质体,将pCas9-miR394载体转入红麻叶片原生质体中进行瞬时表达,测序结果表明:2个靶点Target site 1(T1)和Target site 2(T2)处都发生碱基突变,表明pCas9-miR394载体在红麻原生质体中具有靶向切割的活性。
文摘MicroRNA (miRNA)是植物基因转录与翻译的重要调控因子,miR394参与植物多种抗逆性的调节过程。为深入研究miR394在小桐子抗冷性中的调控机制,利用生物信息学方法对小桐子miR394进行鉴定及靶基因预测,并通过实时荧光定量PCR分析了jcu-miR394及其预测靶基因在低温胁迫下的差异表达特性。结果表明,小桐子中共鉴定到两个miR394基因(jcu-MIR394a, jcu-MIR394b),都可形成稳定的茎环结构,成熟体序列都位于茎环结构的5′端,且都为20 nt (5′-UUGGCAUUCUGUCCACCUCC-3′)。靶基因预测与RT-qPCR表达分析显示,小桐子FBXO6是miR394的潜在靶基因,并可能通过miR394b-FBXO6靶向互作参与小桐子抗冷性过程。本研究结果为开展小桐子miR394参与抗冷性机制研究奠定了基础。
基金supported by National Nature Science Foundation of China(91535201)State Key Laboratory of Wheat and Maize Crop ScienceSouthern Regional Collaborative Innovation Center for Grain and Oil Crops in China
文摘Rice leaf inclination is an important agronomic trait, closely related to plant architecture and yield.Identification of genes controlling leaf inclination would assist in crop improvement. Although various factors,including the plant hormones auxin and brassinosteroids,have been shown to regulate lamina joint development,the role of microRNAs in regulating leaf inclination remains largely unknown. Here, we functionally characterize the role of rice miR394 and its target, LEAF INCLINCATION 4(LC4), which encodes an F-box protein, in the regulation of leaf inclination. We show that miR394 and LC4 work,antagonistically, to regulate leaf lamina joint development and rice architecture, by modulating expansion and elongation of adaxial parenchyma cells. Suppressed expression of miR394, or enhanced expression of LC4,results in enlarged leaf angles, whereas reducing LC4 expression by CRISPR/Cas9 leads to reduced leaf inclination, suggesting LC4 as candidate for use in rice architecture improvement. LC4 interacts with SKP1, a component of the SCF E3 ubiquitin ligase complex, and transcription of both miR394 and LC4 are regulated by auxin. Rice plants with altered expression of miR394 or LC4 have altered auxin responses, indicating that the miR394-LC4 module mediates auxin effects important for determining rice leaf inclination and architecture.
