摘要：

为加速粉果番茄新品种选育,利用CRISPR/Cas9技术编辑SlMYB12基因快速创制粉果番茄新种质。在番茄红色果实调控基因SlMYB12的第一个外显子区域选取2个片段作为靶序列构建CRISPR/Cas9双元表达载体,以红果番茄R18-10C为试验材料,通过农杆菌介导的叶盘转化法进行遗传转化,利用特异引物筛选无转基因成分的纯合突变体植株,并对其相关农艺性状和果实营养品质进行调查分析。经测序检测发现共获得了3种不同变异类型的纯合突变体,均属于碱基缺失导致的移码突变。与野生型红果番茄相比,SlMYB12基因编辑的突变体植株生长正常,植株高度、单果质量、单株产量、果实可溶性固形物含量、番茄红素含量等性状无显著差异,但其成熟果实表现为粉色,果皮中柚皮素查尔酮含量显著降低。综上所述,利用CRISPR/Cas9技术成功编辑番茄MYB12基因,获得了稳定遗传的粉果番茄新种质。

关键词: 番茄, 粉果, SlMYB12, CRISPR/Cas9, 基因编辑

Abstract:

In order to accelerate the breeding of pink-tomato varieties,a rapid breeding strategy was used to generate pink-fruited tomato material by CRISPR/Cas9-mediated gene editing of SlMYB12.Two adjacent target sites within the first exon of SlMYB12 were selected to construct the CRISPR/Cas9 binary vector, and then the vector was introduced into red-fruited inbred line R18-10C through Agrobacterium-mediated transformation. The homozygous mutants without exogenous Cas9 were screened using specific primers and their agronomic traits and fruit nutritional quality were analyzed. Sequencing results showed that three homozygous mutants of different mutation types were obtained and all of which were frame shift mutations caused by base deletion. Compared with wild-type red fruit tomatoes, SlMYB12-edited plants grew and developed normally,and there were no significant difference in plant height,single-fruit weight,total yield per plant,fruit total soluble solid content and lycopene level,but the mature fruit showed pink and the flavonoid naringenin chalcone(NarCh)content of tomato peels was significantly reduced in MYB12-edited plants.In summary,the procedure for the generation of pink-fruited tomato plants through CRISPR/Cas9-mediated targeted mutagenesis of SlMYB12 was set up and new pink-fruited tomato germplasms with stable inheritance were obtained.

Key words: Tomato, Pink fruit, SlMYB12, CRISPR/Cas9, Gene editing