Objective: To clone the downstream key enzyme IrCYP71 gene in diterpenoids biosynthesis of Isodon rubescens for sequence analysis

carry out prokaryotic expression analysis and subcellular localization for the protein encoded by this gene

and optimize the conditions for protein expression in host cells. Method:The full-length cDNA sequence was cloned according to the IrCYP71 gene fragment obtained from transcriptome sequencing; the recombinant plasmid of pET28a (+) -IrCYP71 was constructed and transformed into Rosetta receptive cells

small amount and large amount expressed proteins before identification. The inclusion body proteins were purified and renaturated

and then the renaturated proteins were purified

identified and analyzed. refolding of inclusion body protein

and analyzed the purification and identification of complex protein.The vector PCR8/GW/TOPO-IrCYP71 was constructed by gateway cloning technology

recombined with transformed Pearleygate104 vector

and then introduced into tobacco epidermal cells by agrobacterium-mediated(GV3101) transformation for protein subcellular localization. Result: The full-length cDNA of cloned IrCYP71 gene was 1 593 bp

encoding 530 amino acids

whichwas registered in GeBank(Accession No.MG800628).The recombinant protein expressed via Escherichia coli showed relatively correct molecular weight

about 62 kDa.The total amount of purified recombinant protein was 1 mg and the protein purity was 85%. Green fluorescence was tested and targeted to nucleus under a laser scanning confocal microscope. Conclusion:The preliminary validation of IrCYP71 gene in I.rubescens revealed the prokaryotic expression and subcellular localization of the expressed proteins

laying foundation for further elucidating the function of the gene inditerpenoidsbiosynthesis.