牛樟芝FAD2-1、FAD2-2和SCD基因异源表达及其蛋白互作OA

[Objective]The heterologous expression and protein interaction of FAD2-1,FAD2-2 and SCD genes in Antrodia cinnamomea were investigated to lay the foundation for research and application of metabolic pathways of A.cinnamomea fat acid.[Method]The fusion gene expression vector,protein purification,protein primary structure function and yeast two-hybrid were built by heterologous expression of FAD2-1,FAD2-2 and SCD genes expressed by the expression system of a baculovirus-insect cell to study the effect of oleic acid and linoleic acid exogenous additives on mycelial growing development of A.cinnamomea.[Result]The size of FAD2-1,FAD2-2 and SCD proteins was 40 kD,20 kD and 60 kD,respectively.The expression quantity of FAD2-2 protein was the highest.The protein spectrum primary structure analysis showed that FAD2-2 protein possessed methylation,ubiquitination,carbamylation and 4-hydroxynonenal adduction modification,which revealed that FAD2-2 had a higher interaction possibility with other proteins.The molecular structure of FAD2-1 and SCD proteins was stable.The yeast two-hybrid analysis showed that FAD2-2 interacted with SCD to a certain extent,and the interaction might improve its metabolism.Adding linoleic acid had the significant promoting effect on mycelium growth characteristics(mycelium growth rate and biomass accumulation rate)of A.cinnamomea,and the mycelium growth rate of A.cinnamomea under adding 0.11 μg/mL of linoleic acid reached 1.238 mm/d.[Conclusion]The heterologous expression and recombinant proteins purification of three key genes(FAD2-1,FAD2-2,and SCD)related to fat acid metabolism in A.cinnamomea are successfully implemented by using a baculovirus-insect cell expression system.FAD2-1 is predominantly modified by oxidation,and FAD2-2 can be modified by multiple modification types of methylation and 4-hydroxynonenal adduction(4-HNE).SCD can be modified by cysteine carbamido methylation.The interactions between SCD and FAD2-1,FAD2-2 may synergistically improve the metabolic flux of oleic and linoleic acids in A.cinnamomea.Adding exogenous linoleic acid can promote mycelium growth of A.cinnamomea significantly.