CRISPR/Cas9系统在本氏烟草基因敲除中的应用

The Application of CRISPR/Cas9 System in Gene Knock-out of Nicotiana benthamiana

CRISPR(clustered regulatory interspersed short palindromic repeat)/Cas9(CRISPR associated proteins)是源于原核生物的一种获得性免疫系统,广泛应用于细菌、真菌、动物和植物的基因编辑研究中。本研究是以转基因本氏烟草16C(16C transgenic Nicotiana benthamiana,16C-NB)为材料,利用CRISPR/Cas9技术实现对其GFP(green fluorescent protein)基因的编辑工作,使用PCR-RE(polymerase chain reaction-restriction enzyme)的方法检测了突变体,建立了本氏烟草CRISPR/Cas9基因敲除瞬时表达系统,进而探讨了CRISPR/Cas9系统在植物基因敲除中的应用。在本研究中,采用In-Fusion克隆技术获得了操作性强的中间载体,针对靶基因GFP设计特定靶位点并进行了基因突变实验,为将来开展其它植物内源基因的生物学功能研究提供了参考。

CRISPR（clustered regulatory interspersed short palindromic repeat）/Cas9（CRISPR associated proteins）is an adaptive immune system in prokaryotic organisms, which is widely used in gene editing studies on bacteria,yeast, animals and plants. This research chose the 16 C transgenic Nicotiana benthamiana（16C-NB） as material,edited its GFP（green fluorescent protein） gene by CRISPR /Cas9 technique, detected the mutants by PCR-RE（polymerase chain reaction-restriction enzyme）, established a transient expression system of CRISPR/Cas9 gene knock-out for Nicotiana benthamiana, and discussed the potential application of CRISPR/Cas9 system in plants’ gene knock-out. In this research, we acquired the intermediate vector with strong operability by In-Fusion cloning technology, designed a specific target site based on the GFP target gene, and did a gene mutation test, which provided references for further studies on biological function of endogenous genes in other plants.