引导编辑介导的水稻HPPD基因原位插入flag标签编码序列的遗传材料创制OA

4-hydroxyphenylpyruvate dioxygenase(HPPD)is a key enzyme in the synthesis pathway of tyrosine to plastoquinone and tocopherol in organisms,and it is also an important target for herbicide.Investigation of transcriptional and translational regulatory mechanisms of HPPD gene is of great significance for improving rice abiotic stress resistance and developing herbicide-resistant rice varieties.In this study,the coding sequence of 3×flag tag was precisely inserted into the 3′ end of rice HPPD gene through prime editing(PE)technology.Three T0 generation plants with precise insertion of the coding sequence of 3×flag tag were identified.Western blot results indicated that the fusion protein of HPPD-3×flag ex-pressed normally.The genotyping results of self-pollinated T1 generation plants indicated that the precise insertion event of one T0 generation plant was inherited to the next generation,and T1 generation homozygous plants harbouring precise inser-tion without transgenic elements were identified.The results of real-time quantitative PCR revealed that at different time points after the transition from dark to light,the transcriptional level of rice HPPD gene exhibited a trend from high to low,while the expression abundance of HPPD protein was comparable at different time points.The rice germplasm expressing HPPD-3×flag fusion protein in situ generated in this research provides an important genetic material for investigating the regulatory mechanisms of rice HPPD gene at translational and post-translational levels.