Preliminary Analysis Of β-carotene Synthesis Inducement By Light Through Blue Light Receptors In Blakeslea Trispora

β-carotene is a natural pigment that is widely used in food,medicine,cosmetics and other fields,and can currently be industrially produced by Blakeslea trispora.The synthesis of β-carotene by B.trispora is regulated by blue light.This process is mediated by its blue light receptors BTWC-1(A,B,C)and BTWC-2(A,B,C,D).Although it has been confirmed in some filamentous fungi that blue light receptors can play a regulatory role in carotenoid synthesis by forming complexes as transcription factors to bind to carotenoid structural gene promoters,there are still few studies on the regulation of blue light receptors in B.trispora β-carotene synthesis,which is not conducive to elucidating the light-induced β-carotene synthesis mechanism in B.trispora.In view of this research status,this study carried out a preliminary analysis of the β-carotene synthesis regulated by the blue light receptor of B.trispora by photoinduced gene expression correlation analysis,bioinformatics analysis,protein interaction analysis,RNA interference and other methods,and the main research contents are as follows.(1)The gene transcription level of blue light receptor and β-carotene structural gene under different illumination modes was analyzed.Different illumination modes were set,and the relative transcription levels of btwc-1(a,b,c)and btwc-2(a,b,c,d)and car B and car RA were determined by real-time PCR.The experimental results show that blue light has a stronger induction effect on the transcription of the above genes than white light,while red light cannot induce the transcription of the above genes.The higher the light intensity,the higher the transcription level of the above genes,and the faster the transcription rate of light response activation;Short-term blue light exposure is more conducive to the continuous high-level transcription of blue light receptor genes and β-carotene structural genes.(2)The correlation between blue light receptors and β-carotene structural gene transcription under different light modes was analyzed.Three illumination modes were set for continuous low-intensity blue light,continuous high-intensity blue light,and short-term high-intensity blue light,and the correlation between three btwc-1 and four btwc-2 and the relative transcription levels of car B and car RA was obtained by Spearman correlation analysis.The results showed that under the condition of continuous low-intensity blue light,only btwc-1a and btwc-1c were highly correlated with the relative transcription levels of car B and car RA.The relative transcription levels of btwc-1(a,b,c)and btwc-2(a,b,c,d)and car B and car RA were significantly or highly correlated except for btwc-2c.(3)Subcellular localization analysis of blue light receptors was performed.By constructing subcellular localization recombinant vectors of BTWC-1 and BTWC-2 fusion red fluorescent protein m Cherry,the transformed strain was obtained by transforming the B.trispora based on the method mediated by Agrobacterium tumefaciens,and a small amount of BTWC-1 fusion red fluorescent protein was localized in the nucleus and cytoplasm,and BTWC-2 fusion red fluorescent protein was localized in the cytoplasm and nucleus at the same time.(4)Interaction analysis between blue light receptors.BTWC-1(A,B,C)and BTWC-2(A,B,C,D)proteins were expressed and purified in Escherichia coli,and the interaction between12 groups of BTWC-1 and BTWC-2 was analyzed by GST-pull down assay.The experimental results show that BTWC-1C can interact with BTWC-2A to form a complex,while the remaining 11 groups of BTWC-1 and BTWC-2 cannot combine with each other.(5)The effect of inhibition of btwc-2a expression on carotenoid synthesis was investigated.Based on the previous study of the significant effects of inhibition of btwc-1c expression on carotenoid synthesis in this laboratory,the role of btwc-2a in the synthesis of β-carotene in the B.trispora was further analyzed.The RNA interference and control vector of btwc-2a was constructed,and the transformation of B.trispora was based on the A.tumefaciens mediated method,and real-time PCR analysis showed that the interference efficiency was 66.7%.The interfering strain and the control strain were cultured under continuous dark and short-term blue light conditions,and the relative transcription levels of β carotene accumulation and carotene and car B and car RA were determined at different times.The results showed that inhibition of btwc-2a expression under dark conditions had no effect on β-carotene accumulation and car B and car RA transcription levels.Inhibition of btwc-2a expression under short-term blue light irradiation reduced the relative transcription levels of car B and car RA by 41.9% and 41.0%,respectively,and the final accumulation of β-carotene by 12.4%.The above results showed that btwc-2a mediated light-induced β-carotene synthesis of B.trispora and was positively regulated.