烟草液泡pH相关基因NtPH1的CRISPR/Cas9敲除载体的构建

Knockout Vector Construction of Tobacco Vacuolar pH-Related NtPH1 Gene by Plant CRISPR/Cas9 System

摘要: 植物液泡pH的碱性程度是蓝色花形成的一个重要因素。烟草NtPH1是矮牵牛蓝色花形成相关的控制液泡pH的PhPH1基因的直系同源基因。为了研究其功能，本研究利用植物CRISPR/Cas9系统构建NtPH1的双靶点敲除载体。通过对NtPH1基因片段的序列分析，选择两个PAM序列相距60 bp的位点作为靶序列，通过重叠PCR直接把两个靶位点分别拼接到AtU6-29和AtU6-26两个启动子驱动的sgRNA表达盒上，再利用Golden Gate克隆的方法把其连入pYLCRISPR/Cas9Pubi-H载体，重组克隆经菌落PCR筛选、质粒DNA酶切鉴定与测序，获得了NtPH1基因的双靶点敲除载体pCas9-PH1T1T2。这为进一步开展NtPH1基因的功能研究与蓝色花的基因工程应用奠定了基础。

Abstract: The alkaline pH level of plant vacuoles is an important factor for coloration of blue flowers. Tobacco NtPH1 gene is an orthologous gene of PhPH1 that controls the vacuolar pH in petunia petals. To study the function of NtPH1 gene, we constructed a knockout vector with two target sites of NtPH1 gene using plant CRISPR/Cas9 System. The two target sequences in NtPH1 genomic DNA are a 60 bp apart. PCR amplified the small nuclear RNA promoters (AtU6-26 and AtU6-29) and two sgRNAs, respectively. For each sgRNA cassette, the overlapping sequence between promoter and sgRNA was the target sequence of 20 bp. The overlapping PCR was completed to in vitro splice the two sgRNA cassettes. Then the two sgRNA cassettes were cloned into pYLCRISPR/Cas9Pubi-H vector by Golden Gate cloning. After positive colony PCR, restriction enzyme digestion and further sequencing, the knockout vector, pCas9-PH1T1T2, was successfully constructed. This work laid a foundation for further study on the function of NtPH1 gene, and future application of this gene in genetic engineering of blue flowers.