丝瓜LcPPO1基因CRISPR/Cas9基因编辑载体的构建  

Construction of CRISPR/Cas9 Gene Editing Vector of LcPPO1 Gene in Luffa

作　　者：陈敏氡[1] 王彬[1] 刘建汀 叶新如 曾美娟[1] 朱海生[1] 温庆放[1] 康玉妹[1] CHEN Min-dong;WANG Bin;LIU Jian-ting(Fujian Key Laboratory of Vegetable Genetics and Breeding,Crops Research Institute,Fujian Academy of Agricultural Sciences,Vegetable Research Center,Fujian Engineering Research Center for Vegetables,Fuzhou,Fujian 350013)

机构地区：[1]福建省蔬菜遗传育种重点实验室,福建省农业科学院作物研究所,蔬菜研究中心/福建省蔬菜工程技术研究中心,福建福州350013

出　　处：《安徽农业科学》2021年第3期105-109,共5页Journal of Anhui Agricultural Sciences

基　　金：福建省自然科学基金项目(2019J01112);福建省现代蔬菜产业技术体系岗位专家项目(2018R1026-5);福建省农业科学院科技创新团队建设项目(STIT2017-1-2);国家大宗蔬菜产业技术体系福州综合试验站项目(CARS-23-G-53);设施蔬菜种质收集及基因发掘、品种创新与产业化(2018NZ0002-3);福建省属公益类专项(2019R1031-2)。

摘　　要：[目的]通过CRISPR/Cas9系统构建丝瓜LcPPO1基因编辑载体。[方法]根据前期已经克隆得到的LcPPO1基因序列,设计2个sgRNA靶位点序列,退火制备sgRNA双链,分别与线性PCA1301-Cas9载体连接获得2个重组载体,将重组载体转化DH5α感受态细胞,再对其进行PCR鉴定及测序比对分析。[结果]2个sgRNA靶位点序列已经分别准确地连入PCA1301-Cas9载体,插入序列无突变。[结论]成功构建了丝瓜LcPPO1基因编辑载体PCA1301-Cas9-sgRNA1和PCA1301-Cas9-sgRNA2,为后续研究丝瓜LcPPO1基因功能奠定了基础。[Objective]LcPPO1 gene editing vector for luffa was constructed by CRISPR/Cas9 system.[Method]Based on the sequence of LcPPO1 gene cloned in previous stage,two sgRNA target sequences were designed,and the sgRNA double strand was prepared by annealing.Then the sgRNA double strands were connected with the linear PCA1301-Cas9 vector to obtain two recombinant vectors,respectively.The recombinant vectors were transformed into E.coli DH5α,and then identified by PCR and sequenced.[Result]Two sgRNA target sequences were accurately linked into the PCA1301-Cas9 vector,respectively,and the insertion sequence had no mutation.[Conclusion]Two LcPPO1 gene editing vectors for luffa,PCA1301-Cas9-sgRNA1 and PCA1301-Cas9-sgRNA2,were successfully constructed,which laid a foundation for further study on the function of LcPPO1 gene in luffa.

关 键 词：丝瓜 LcPPO1基因 CRISPR/Cas9技术 基因编辑 

分 类 号：S188[农业科学—农业基础科学]