基于CRISPR/Cas9的月季TCP9基因融合表达载体构建

Construction of Rose TCP9 Gene Fusion Expression Vector Based on CRISPR/Cas9

CRISPR/Cas9基因编辑系统是植物基因功能研究的重要工具,月季TCP9基因可以调控花器官的发育,应用CRISPR/Cas9技术构建敲除月季TCP9基因的表达载体,可以为月季TCP9基因的调控机制提供更多信息。根据TCP9基因的保守序列,设计了一对互补引物,用T4连接酶连接到质粒载体pKSE402 (该载体含有eGFP)中;通过测序确定所构建载体的正确性,通过冻融法将含有TCP9基因的重组质粒转入农杆菌(EHA105)中。以农杆菌为模板PCR扩增以及eGFP瞬时表达证明月季TCP9基因敲除表达载体已构建成功。本研究表明了含有TCP9基因的表达载体能在植物中高效表达,为进一步研究提供了一定的科学依据。

CRISPR/Cas9 gene editing system is an important tool for plant gene function research.Rose TCP9 gene can regulate floral organ development.CRISPR/Cas9 technology is applied to construct the knockout vector of TCP9 gene,which can provide more information for the regulation mechanism of TCP9 gene in rose.According to the conservative sequence of TCP9 gene,a pair of complementary primers was designed and was connected to the plasmid vector pkSE402 by T4 ligase containing eGFP;The correctness of the constructed vector was confirmed by sequencing,and the recombinant plasmid containing TCP9 gene was transferred into Agrobacterium tumefaciens(EHA105)by freeze-thaw method.PCR amplification using Agrobacterium tumefaciens as template and transient expression of eGFP proved that the expression vector of TCP9 gene knockout in Rose had been constructed successfully.The study indicates that the expression vector containing TCP9 gene could be efficiently expressed in plants,providing a scientific basis for further research.