利用CRISPR/Cas9技术定向编辑加工番茄SlACS2

Targeted Editing ofSlACS2by CRISPR/Cas9 Technology in Tomato

加工番茄为呼吸跃变型果实,伴随呼吸跃变产生大量乙烯,即系统Ⅱ乙烯,易使番茄果实过熟,腐烂变质。SlACS2是番茄系统Ⅱ乙烯合成的限速酶,旨在通过CRISPR-Cas9基因组编辑系统修饰该基因,调控系统Ⅱ乙烯过量表达,以迟滞番茄过熟腐烂。利用CRISPR/Cas9系统定点编辑加工番茄SlACS2基因,在SlACS2的第2外显子区域设计2个靶位点,构建双靶点的CRISPR/Cas9敲除载体,通过农杆菌介导法转化加工番茄,再生培养获得T0代转基因幼苗,通过PCR扩增卡那霉素抗性基因获得阳性株系。为进一步获得纯合突变,对T1代植株的双靶位点区域进行PCR扩增和测序分析,鉴定SlACS2突变类型。结果发现,从阳性植株的T1后代中鉴定出6种在两个靶位点发生纯合突变类型植株,其中靶位点1突变类型较为丰富,分别发生单碱基的插入及1个、4个、5个和9个碱基的缺失;靶位点2则只有7个碱基的缺失一种编辑类型。结果表明,已成功在加工番茄体内实现对内源SlACS2的定点敲除,获得的基因编辑植株,为进一步筛选耐贮突变体提供材料基础。

Processed tomato is typical climacteric fruit.With the climacteric of tomato,tomato can produce large amounts of ethylene,i.e.systemⅡethylene,which causes overripe and rot.ACC synthase 2 in tomato(SlACS2)is the key enzymy during the process of biosynthesis of the systemⅡethylene.The expression of the systemⅡethylene was regulated to delay the fruits to overripen.The CRISPR/Cas9 genome editing system was used to edit and modofy theSlACS2genes of tomato.Two target sites were designed in the second exon region ofSlACS2,and a double-target CRISPR/Cas9 knockout vector was constructed.Tomato was transformed and processed by Agrobacterium tumefaciens mediated method,and T0 generation of transgenic seedlings were obtained by regeneration culture.Kanamycin resistance gene was amplified to obtain positive strains by PCR.To further obtain homozygous mutations,PCR amplification and sequencing analysis were performed on the dual-target region of T1 generation plants to identifySlACS2mutation types.The results found that six homozygous mutations in two targeted point type plants were identified from the T1 progeny of the positive plants,of which targeted point mutation type 1 was relatively rich,single base insertion and deletion of 1,4,5 and 9 bases occurred,respectively.Only one editing type with 7 base missed in target site 2.The results showed that the targeted deletion of endogenousSlACS2was successfully achieved in processed tomato,and gene-edited plants provide material for further screening of storage-tolerant mutants.