【摘要】 为培育成熟果实软化程度低,货架期长的番茄植株,以加工番茄甘露糖苷酶基因(α-Man)为编辑对象,设计由番茄U6启动子驱动、长21 bp的guide RNA(gRNA)指导hCas9核酸酶,靶向编辑α-Man的第1个外显子。首先构建基于CRISPR/Cas9系统的植物表达载体,并通过农杆菌介导的遗传转化获得加工番茄转基因株系,然后取转基因番茄叶片基因组DNA,利用限制性内切酶法结合PCR扩增对α-Man编辑位点附近的DNA片段进行检测及测序分析。结果表明,14株转基因番茄植株有2株检测到突变现象。α-Man突变体TA克隆测序结果显示有2种编辑类型,一种表现为52 bp的缺失突变;另一种表现为单碱基突变。实现了对番茄α-Man的编辑。更多还原

【Abstract】 In order to cultivate tomato variety with low-softening-degree and long-shelf-duration fruit,the first exon of the N-glycan processing enzyme gene(α-Man)was edited directly by the hCas9 nuclease combined with 21 bp guide RNA(gRNA)and while driven by tomato U6 promoter. Firstly,the plant expression vector based on CRISPR/Cas9 system was constructed,and the transgenic lines of tomato were obtained by Agrobacterium-mediated genetic transformation. Then the DNA fragment near α-Man’s editing site from the genomic DNA of transgenic tomato leaves was detected and sequenced by restriction endonuclease and PCR amplification. The results showed that 2 of 14 transgenic tomato strains were detected to have mutations. The sequencing results of the α-Man mutant TA clone suggested that there were 2 types of editing,one was a 52 bp deletion mutation and the other was a single base mutation. This indicates that the editing of tomato α-Man gene is achieved.更多还原