【摘要】 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。更多还原

【Abstract】 In order to explore the function of miRNA in kenaf,based on the results of high throughput miRNA sequencing,miR394,a microRNA,precursor gene sequence of kenaf was cloned using kenaf genomic DNA as template.The result showed that the sequence size of pre-miR394 of kenaf was 175 bp,and the precursor sequence formed a stable secondary structure analyzed using Mfold software.A reliable CRISPR/Cas9 target sequence was designed according to the miR394 precursor gene sequence of kenaf.In this study,taking the AtU3 band AtU6-29 plasmids as templates and based on an improved CRISPR/Cas9 multi-target vector system,AtU3 b-sgRNA and AtU6-29-sgRNA expression cassettes were successfully constructed using overlapping PCR technique.The two expression cassettes were successfully constructed into pYLCRISPR/Cas9 vectors according to Golden Gate Cloning method.The constructed vector was named pCas9-miR394.pCas9-miR394 was then transiently expressed in the protoplasts of kenaf leaves.The sequencing result indicated that one base mutation occurred at Target site 1(T1)and Target site2(T2),demonstrating that the pCas9-miR394 vector had target cutting activity in kenaf protoplasts.This vector could be used for subsequent genetic transformation analysis in kenaf.更多还原