棉花U3和U6启动子在CRISPR/Cas9基因组编辑体系中的功能鉴定

Functional Identification of Cotton U3 and U6 Promoters in the CRISPR/Cas9 Genome Editing System

【目的】对已克隆的海岛棉U3和U6启动子进行功能鉴定,为构建棉花CRISPR/Cas9多位点基因编辑技术体系提供更多可用的U3和U6启动子。【方法】分别构建以GbU3-2P和GbU6-7P为启动子驱动sg RNA,以抗旱负调控基因GGB为靶序列的CRISPR/Cas9基因编辑载体,然后在新海16的棉花叶片原生质体中进行功能鉴定。通过Polymerase chain reaction方法富集构建好的CRISPR/Cas9基因编辑载体的核心片段,并利用PEG瞬时转化法将核心片段转入原生质体中。提取转化后的原生质体基因组DNA,采用酶切/Polymerase chain reaction法分析棉花GGB基因的突变情况并测序验证。最后绘制靶基因突变的频率分布图来计算该CRISPR/Cas9系统的编辑效率和确认突变的真实性。【结果】靶基因测序结果显示靶标位点序列突变的类型全部为碱基替换。【结论】以GbU3-2P和GbU6-7P为启动子的CRISPR/Cas9基因编辑体系可以成功地定点编辑棉花GGB基因的序列,引起基因突变。

[Objective]The function of U3 and U6 promoters that were cloned from sea-island cotton were identified in order to provide more available U3 and U6 promoters for the construction of cotton CRISPR/Cas9 multi-sites gene editing system.[Method]Two CRISPR/Cas9 gene editing vectors were constructed,in which sgRNA were driven by GbU6-7P and GbU3-2P,respectively,and GGB,a negative regulator in drought tolerance,was used as target gene.The function of the vector were identified in cotton leaf protoplast of Xinhai 16.The core fragment of CRISPR/Cas9 gene editing vector were enriched by Polymerase chain reaction method and were delivered into protoplast through PEG transient transformation.Then genomic DNA were extracted from protoplast.Gene mutations were analyzed using Enzyme digest/Polymerase chain reaction and sequenced.Finaly the mutation efficiencies of the CRISPR/Cas9 system were calculated and the frequency distribution of the mutation in target site were drawn in order to confirm the authenticity of the mutation.[Result]All type of mutation in target loci were base substitution.[Conclusion]Both CRISPR/Cas9 gene editing systems based on GbU3-2P and GbU6-7P promoters could successfully edit the sequence of cotton GGB gene and cause gene mutation.