| Carotenoids are natural pigments with antioxidant effects and are widely used in various industries.Blakeslea trispora is an ideal strain for carotenoid preparation and its synthesis process is regulated by the negative regulator Crg A.Crg A is commonly found in B.trispora and some other filamentous fungis and has ubiquitin ligase activity,whose effect on the regulation of carotenoid synthesis may be mediated through a ubiquitination process.There are few studies on the regulation of carotenoid synthesis by Crg A in B.trispora,especially its function as a ubiquitin ligase has not been reported.In this study,we analyzed the effect of btcrg A on carotenoid synthesis through in vivo RNA interference and overexpression assay.In addition,we determined the ubiquitin ligase function of Bt Crg A by mining the ubiquitinylase catalytic reaction elements and constructing an in vitro enzyme-catalyzed reaction for Bt Crg A of B.trispora,expecting to lay some foundation for elucidating the mechanism of Crg A regulation of carotenoid synthesis,and the details of this study are as follows.1)Correlation analysis of the expression of btcrg A with light and the expression of carotenoid structural gen.The cis-acting element prediction of the promoter sequence of btcrg A showed that it has several light response-related elements,indicating that the transcription of this gene is closely related to light.Darkness,continuous light and short light treatments were set up for B.trispora,and correlation analysis was performed on the transcript levels of btcrg A and car B and car RA.The results showed that the transcriptional levels of the three genes reached the highest level in a short period of time,showing the phenomenon of "light response" and "light adaptation",and the relative transcript levels were higher due to light stimulation.Correlation analysis showed that in continuous light,btcrg A was highly correlated with the expression of car B and lowly correlated with car RA,in short light,btcrg A was highly correlated with the expression of car B and car RA,indicating that btcrg A is related to the regulation of carotenoid synthesis.2)Analysis of the effect of btcrg A on carotenoid synthesis.The effects of btcrg A on carotenoid synthesis in dark and light cultures were initially analyzed by constructing RNA interferencing and overexpressing strains of btcrg A.In the dark culture,the RNA interferencing strain of btcrg A showed a slight increase in the transcript level of car B,a significant increase in the transcript level of car RA,an increase in the enzyme activity of PDS,PSY and LYC,and a 32% increase in carotenoid accumulation,while the overexpressing strain showed no significant change in the transcript level of car B,a significant decrease in the transcript level of car RA,no significant change in the enzyme activity of PDS,the PSY enzyme activity decreased significantly but the LYC enzyme activity increased,and a 26% decrease in carotenoid accumulation.Compared to the control strain,the btcrg A RNA interferencing strain and the overexpressing strain showed higher levels of car B and car RA transcripts,three specific enzyme activities,and carotenoid accumulation in the light culture compared to the dark.This result indicated that Bt Crg A negatively regulated carotenoid synthesis in dark culture,but the elevated carotenoid accumulation in light culture was related to the level of Bt Crg A,which regulated carotenoid synthesis in light,but not negatively.3)Cloning and bioinformatics analysis of ubiquitination-related enzymes from B.trispora.One ubiquitin-activating enzyme E1 and 18 putative ubiquitin-conjugating enzymes E2 were obtained from B.trispora,bioinformatics analysis showed that Bt E1 contained a Ube1 superfamily structure and belonged to ubiquitin-activating enzymes,and all 18 putative ubiquitin-conjugating enzymes E2 contained a UBC conserved structural domain and were UBC proteins,and it was tentatively determined that they all belonged to ubiquitin-conjugating enzymes.4)In vitro ubiquitination analysis of Bt Crg A.A heterologous expression system using Escherichia coli was used to express Bt E1,Bt Crg A Bt WC-1b and 11 Bt E2 s.The in vitro ubiquitination enzyme catalytic reaction assay showed that Bt Crg A could complete its ubiquitin modification function with the assistance of Bt E1 and Bt UBC D.The ubiquitin ligase function of Bt Crg A was determined,but its ubiquitination modification of Bt WC-1b has not been observed.Fixed-point mutations and truncated mutations of several structural domains of Bt Crg A showed that Bt Crg A requires the simultaneous presence of two ring-finger structural domains to function as a ubiquitin ligase. |
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