CRISPR/Cas9介导的拟南芥TT1基因的敲除

在菜油品质上黄籽相较于褐籽更为优良,且更易于合成高质量的生物柴油。已有研究表明TT1(TRANSPARENT TESTA 1)基因是类黄酮代谢途径中的关键基因,为了通过CRISPR/Cas9基因编辑载体验证TT1基因在白菜型油菜中的功能,本研究先在拟南芥中进行了TT1基因的编辑。构建了CRISPR/Cas9 TT1基因编辑载体,通过农杆菌介导侵染拟南芥花序的遗传转化方式将编辑载体引入拟南芥Columbia生态型,通过表型观察发现T1代12株阳性苗中产生了11株表型株,其中1株表现为褐籽,2株表现为黄籽,9株表现为黄籽和褐籽的嵌合。测序检测分析其突变型结果发现,TT1靶位点处有单碱基缺失、单碱基插入、多个碱基缺失、碱基缺失后插入以及两个靶位点之间多个碱基缺失的多种突变类型。T2代获得一株纯合突变体,TT1两个靶位点之间产生79 bp的碱基缺失,且全株表现为黄籽。本研究为CRISPR/Cas9载体在白菜型油菜中TT1基因功能验证提供借鉴,并为其它作物TT1同源基因的功能验证提供了新的突变体材料。

Knockout of TT1 Gene Caused by CRISPR/Cas9 in Arabidopsis thaliana

Yellow-seed rapaseed is superior to brown-seed in oil quality and is easier to synthesize high quality biodiesel. Previous study showed that the TT1(TRANSPARENT TESTA1) gene is a key gene in the flavonoid metabolic pathway. To identify the function of the TT1 gene in Brassica rapa by the CRISPR/Cas9, our first editing TT1 in Arabidopsis thaliana. The TT1 gene editing vector was constructed, and the vector was introduced into the Arabidopsis Columbia ecotype by Agrobacterium-mediated genetic transformation of Arabidopsis inflorescence.The phenotypic observation revealed that T1 generation 12 positive seedlings were produced. There were 11 phenotypic strains, one of which showed brown seed, two of which showed yellow seed, and nine of which showed the mosaic of yellow and brown seeds. Sequencing detection and analysis of the mutant results revealed that there were multiple mutantion at the TT1 target site, including single base deletion and insertion, multiple bases deletion,multiple bases insertion after bases deletion and multiple bases deletion between two target sites. A homozygous mutant was obtained in the T2 generation, and a 79 bp base deletion was found between the two target sites of TT1,and the whole plant was characterized by yellow seeds. This study provides a reference for the CRISPR/Cas9 vector to verify the function of TT1 gene in B. rapa and provides a new mutant material for functional verification of other crop TT1 homologous genes.