基于CRISPR/Cas9系统制作拟南芥糖基转移酶ugt79b2/79b3双突变体纯合株系

Production of Arabidopsis Glycosyltransferase ugt79b2/79b3 Double Mutant Pure and Strain Based on CRISPR/Cas9 System

CRISPR/Cas9系统在植物遗传改良及功能基因研究中有着非常重要的作用。本研究中,根据拟南芥糖基转移酶家族中同工酶UGT79B2/79B3的前体序列,设计针对靶基因UGT79B2/79B3的特异的sgRNA引导序列,构建ugt79b2/79b3-Cas9双突变植物表达载体,并转化到根癌农杆菌GV3101中。将含有目标载体的GV3101活化后,花浸染法浸染拟南芥。以后代阳性株系DNA作为模板,送公司测序。99株阳性转基因株系中分子鉴定发现有24株发生突变,其中只有1株发生ugt79b2/79b3双突变。该研究结果为拟南芥糖基转移酶ugt79b2/79b3突变体的功能开发提供了基础研究与方法支持。

The CRISPR/Cas9 system plays a very important role in plant genetic improvement and functional gene research.In this study,based on the precursor sequence of the isoenzyme UGT79B2/79B3 in the Arabidopsis glycosyltransferase family,a specific sgRNA guide sequence for the target gene UGT79B2/79B3 was designed to construct a ugt79b2/79b3-Cas9 double mutant plant expression vector,and transformed into Agrobacterium tumefaciens GV3101.After activating GV3101 containing the target vector,Arabidopsis thaliana wild-type plants were transformed by the flower dip method.The DNA of the subsequent generations of positive strains was used as a template and sent to the company for sequencing.Molecular identification of 99 positive transgenic strains revealed that 24 strains had mutations,of which only one strain had ugt79b2/79b3 double mutation.The results of this study provide basic research and method support for the functional development of Arabidopsis glycosyltransferase ugt79b2/79b3 mutants.