应用CRISPR/Cas9体系对番茄PSY 1基因的定点编辑

基于NCBI数据库提供的番茄八氢番茄红素合成酶基因(PSY 1)全长,并根据基因序列中PAM(proto adjacent motif)的位置设计特异性sg RNA,构建CRISPR/Cas9 Level 1、Level 2载体;将设计好的载体转化农杆菌,继而转染番茄子叶,利用PCR产物测序法检测sg RNA在番茄基因组中的敲除效率。结果表明,36个转基因植株中有22个PSY 1基因目标区出现不同数目的碱基缺失、增加或互换,初步估计基因敲除效率为61%。本试验在番茄中初步建立了CRISPR/Cas9介导的基因敲除技术,该技术能够稳定的在番茄中实现基因敲除。

Targeting Modification PSY 1 Gene in Tomato Using CRISPR/Cas9 System

Based on the full length of eight hydrogen phytoene synthase（PSY 1）from NCBI databases， this study designed specific sgRNA according to the PAM（proto adjacent motif）site of the gene sequence， and constructed CRISPR/Cas9 Level 1 and Level 2 vectors. Then the designed vector was transformed to Agrobacterium，by the Level 2 vector were used to transform the tomato cotyledons. The knockout efficiency of sgRNA in tomato genome was detected by PCR product sequencing. The results showed that 22 of the 36 transgenic plants showed a different number of base deletion，increase or interchanges in the targeting area of PSY 1. The gene knockout efficiency was 61% by initial estimates. The technique of CRISPR/Cas9 mediated gene knockout was established in this study. This technique could stably generate gene knockout in tomato.