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目前CRISPR/Cas9技术作为一种基因组定点编辑技术已经被广泛应用于不同植物中,但是对于实现特定碱基替换仍具有很大难度.本研究将APOBEC1和UGI与Cas9n融合,开发了一种包含rBE3和rBE4的高效技术,并通过该技术对OsSERK1,OsSERK2,ipa1,pi-ta和OsBRI1等基因的靶位点碱基进行编辑.在T0代转基因植物中rBE3/sgRNA将胞嘧啶碱基转换为所有其他核苷酸碱基中效率高达38.9%;利用rBE4还获得了抗稻瘟基因pi-ta的隐性等位基因靶位点胞嘧啶碱基替换为胸腺嘧啶碱基,编辑效率达18.2%.此外,在所检测的材料中,并未检测到由Cas9n切口酶引起的InDel事件.这些结果表明,rBE3和rBE4技术的开发,在未来植物基础研究和农业应用中具有广阔的前景.
At present, CRISPR / Cas9 technology has been widely used in different plants as a genome-specific editing technique, but it is still very difficult to achieve specific base replacement.In this study, we fused APOBEC1 with UGI with Cas9n and developed a novel vector containing rBE3 And rBE4 and through which the base sites for the target sites of genes such as OsSERK1, OsSERK2, ipa1, pi-ta, and OsBRI1 are edited. The rBE3 / sgRNA in T0 transgenic plants converts cytosine bases into all The efficiency of other nucleotide bases was as high as 38.9%. The rBE4 was also used to replace the thymine base of the recessive allele target site of the pi-ta gene of blast-resistance gene with an editing efficiency of 18.2%. In addition, no InDel event caused by Cas9n nickase was detected in the tested materials.These results indicate that the development of rBE3 and rBE4 technologies has broad prospects in future plant basic research and agricultural applications.