CRISPR/Cas9系统介导的棉花GhNAC3基因编辑

CRISPR/Cas9 Targeted Editing of GhNAC3 in Cotton

为创制棉花耐旱种质资源,解决棉花耐旱资源贫乏以及提高水资源利用率,研究依据CRISPR/Cas9编辑原理,对课题组前期利用RT-PCR技术筛选耐旱相关基因GhNAC3(Gh_D02G0790)的第一外显子区域设计2个20 bp的编辑靶点,并在陆地棉基因组数据库中比对分析靶点序列,排除非特异性编辑,将2个靶点核苷酸片段分别与gRNA-AtU6载体连接,通过2次PCR扩增,得到含特异性连接接头的AtU6-GhNAC3表达盒,再将表达盒连接到CRISPR/Cas9(pRGEB32-7)载体上,获得CRISPR-GhNAC3重组表达载体,利用农杆菌介导法转化陆地棉受体YZ-1,再生培养得到T_(0)代转基因幼苗,通过PCR检测Cas9蛋白基因获得阳性株系。对T_(0)代植株的靶点区域序列进行PCR扩增和测序分析,鉴定GhNAC3编辑类型。结果发现,CRISPR9-GhNAC3表达载体成功转化YZ-1,并获得40株转基因再生植株,经Cas9蛋白基因鉴定得到30株阳性株系,从阳性植株选择10株进行编辑类型测序分析,发现7株在靶点区域发生编辑,编辑类型主要为碱基片段缺失,缺失片段大小为3~28 bp,T_(1)代种植于大田,获得了同时出现黄化和矮化的突变体3株。

The effective way to address the scarcity of cotton drought resistance resources and improve water resource utilization is exploiting and utilizing key regulatory factors for drought resistance and cultivating drought resistant cotton varieties.According to the principle of CRISPR/Cas9 technology,two 20 bp targeted sequences were designed in the first exon of the GhNAC3 genome sequence.The gene shuffling of non-specific target sites was eliminated using the blast analysis.The oligonucleotides of two target sites were inserted into the gRNA-AtU6 plasmid vector,and then were amplified twice by PCR technology to construct AtU6-GhNAC3 expression-boxes.The recombinant CRISPR-GhNAC3 vector was obtained by linking two expression-boxes to CRISPR/Cas9(pRGEB32-7)vector.The CRISPR-GhNAC3 vector was transformed into the cotton of YZ-1 through agrobacterium mediated method.The T_(0) generation of transgenic plants was obtained by detection of Cas9 protein gene,and the positive transgenic plants were selected through PCR method.The target sites of each T_(0) generation from were determined via PCR and sequencing method,and finally the mutated genotypes were identified.After Cas9 protein gene identification,30 positive strains were obtained,and 10 of them were selected from the positive plants for editing type sequencing analysis.It was found that 7 strains underwent editing in the target region.The editing type was mainly base fragment deletion,with a deletion fragment size of 3-28 bp.T_(1) was planted in the field,and 3 mutants with both yellowing and dwarfing were obtained.