靶向加工番茄elF4E1的CRISPR/Cas9载体有效性验证

【目的】验证基于CRISPR/Cas9系统构建的靶向编辑加工番茄(Solanum lycopersicum) eIF4E1基因载体的有效性,为CRISPR/Cas9系统在培育PVY抗性植株中的应用提供技术支持。【方法】构建靶向编辑番茄真核翻译起始因子elF4E1基因的CRISPR/Cas9系统表达载体,用农杆菌渗透法瞬时转化番茄植株,PCR扩增已转化植株靶位点周围DNA序列后用HaeⅢ进行酶切,回收未切开的条带与pGEM-T载体连接后进行单克隆测序。【结果】对测得的9个克隆序列进行比对分析,在PAM (protospacer adjacent motifs)上游的6~8 bp的碱基处均发生突变,并且都为单碱基的替换,导致多肽链中单个氨基酸的替换。【结论】利用CRISPR/Cas9基因组编辑系统构建的载体能够特异性地靶向加工番茄eIF4E1基因,为利用CRISPR/Cas9系统敲除eIF4E1基因,获得抗PVY病毒的番茄育种材料奠定了基础。

Verification of the Effectiveness of CRISPR/Cas9 Vectors Targeting to Processing Tomato eIF4E1

【Objective】 To verify the effectiveness of eIF4E1 gene vector constructed based on CRISPR/Cas9 system for targeted genome editing processing tomato (Solanum lycopersicum) and to provide technical support for the application of CRISPR/Cas9 system in the cultivation of PVY resistant plants.【Method】Constructing a CRISPR/Cas9 system expression vector targeting to the elF4E1 gene of tomato eukaryotic translation initiation factor, the tomato plants were transiently transformed by Agrobacterium tumefaciens infiltration method. The DNA sequences around the target sites of the transformed plants were amplified by PCR and digested with Hae III, the bands that had not been successfully digested were recovered and ligated with pGEM-T vector for monoclonal sequencing.【Result】An alignment analysis of the 9 cloned sequences revealed that mutations occurred at 6-8 bp upstream of the PAM (protospacer adjacent motifs), and they were all single-base substitutions, resulting in the replacement of a single amino acid in the polypeptide chain.【Conclusion】In this study, the vector constructed by CRISPR/Cas9 genome editing system can specifically target the tomato eIF4E1 gene, which has laid the foundation for the subsequent use of the CRISPR/Cas9 system to knock out the eIF4E1 gene and obtain the tomato breeding materials against PVY virus.