The photoinduced β-carotene synthesis in Blakeslea trispora is dependent on WC-2A

Abstract

β-Carotene, a high value-added natural pigment, is currently produced industrially in Blakeslea trispora. Although photoinduced carotenoid synthesis has been identified in some filamentous fungi, there are still relatively few studies focusing on B. trispora and its potential mechanisms. In this study, an integrated strategy-including correlation analysis of gene expression, bioinformatics analysis, protein interaction, and RNA interference-was adopted to elucidate photoinduced β-carotene synthesis in B. trispora. Light wavelength, intensity, and irradiation duration stimulated the transcription of photoreceptors [btwc-1 (a, b, c) and btwc-2 (a, b, c, d)] and carotenoid structural genes (carB and carRA). The transcription of photoreceptor genes showed significant or high correlation with carotenoid structural genes under continuous or short-term, high-intensity blue light irradiation. To elucidate the role of photoreceptors in carotenoid synthesis, the interaction between BTWC-1 and BTWC-2 was predicted. Furthermore, Glutathione S-Transferase (GST) pull-down assays showed that only BTWC-1C and BTWC-2A could interact to form complexes. Inhibition of btwc-2a expression under dark conditions did not affect β-carotene accumulation or the transcription of carB and carRA, but did reduce these parameters under blue light irradiation, indicating that btwc-2a mediates photoinduced β-carotene synthesis in B. trispora.

Keywords: Blakeslea trispora; GST pull-down; RNA interference; photoreceptor; β-carotene.

Figure 1

Relative transcription levels of btwc-1, btwc-2, and carotene structural genes under irradiation of different wavelength light at different times. (A) btwc-1a. (B) btwc-1b. (C) btwc-1c. (D) btwc-2a. (E) btwc-2b. (F) btwc-2c. (G) btwc-2d. (H) carB. (I) carRA. Black, blue, white, and red denote that strains were cultured in the darkness, blue light, white light, and red light, respectively. All strains were pre-cultured in the darkness for 3 days, at which time the transcription levels of these genes were used as controls. Three biological replicates were conducted for each experiment.

Figure 2

Relative transcription levels of btwc-1, btwc-2, and carotene structural genes under blue light irradiation of different intensities at different times. (A) btwc-1a. (B) btwc-1b. (C) btwc-1c. (D) btwc-2a. (E) btwc-2b. (F) btwc-2c. (G) btwc-2d. (H) carB. (I) carRA. All strains were pre-cultured in the dark for 3 days, at which time the transcription levels of these genes were used as controls. Three biological replicates were conducted for each experiment.

Figure 3

Relative transcription levels of btwc-1, btwc-2, and carotene structural genes under continuous (360 min) or short-term (30 min) blue light (1,000 lux) irradiation at different times. (A) btwc-1a. (B) btwc-1b. (C) btwc-1c. (D) btwc-2a. (E) btwc-2b. (F) btwc-2c. (G) btwc-2d. (H) carB. (I) carRA. Black and blue denote that strains were cultured in the darkness and blue light, respectively. Black-blue-black represents strains were exposed to blue light (1,000 lux) for 30 min and then moved into darkness. All strains were pre-cultured in the darkness for 3 days, at which time the transcription levels of these genes were used as controls. Three biological replicates were conducted for each experiment.

Figure 4

SDS-PAGE profiling of crude and purified BTWC-1 and BTWC-2 proteins (in square frames). M is Maker in Figures; (A), lanes 1–9 are: pColdII control, BTWC-1A crude protein, BTWC-1A purified protein, pET-28a control, BTWC-1B crude protein, BTWC-1B purified protein, pColdII control, BTWC-1C crude protein, BTWC-1C purified protein, respectively. (B), lanes 1–9 are: pET-28a control, BTWC-2A crude protein, BTWC-2A purified protein, BTWC-2B crude protein, BTWC-2B purified protein, BTWC-2C crude protein, BTWC-2C purified protein, BTWC-2D crude protein, BTWC-2D purified protein, respectively.

Figure 5

GST Pull down assay of the interaction between 12 sets of BTWC-1 and BTWC-2. In (A), lanes 1–6 are BTWC-1A target protein positive control, GST negative control, HIS-BTWC-1Aeluted bands after co-incubation with GST-BTWC-2A, GST-BTWC-2B, GST-BTWC-2C, and GST-BTWC-2D, respectively; in (B), lanes 1–6 are the positive control for BTWC-1B target protein, the positive control for HIS-BTWC-1B with GST-BTWC-2A, GST-BTWC-2B, GST BTWC-2A, GST-BTWC-2B, GST-BTWC-2B, GST-BTWC-2D, respectively; in (C), lanes 1–6 are the positive control for BTWC-1C target protein, the GST negative control, the elution bands after co-incubation of HIS-BTWC-1C with GST-BTWC-2A, GST-BTWC-2B, GST-BTWC-2C, and GST-BTWC-2D, respectively.

Figure 6

Relative transcription levels of btwc-2a, carotene structural genes, and β-carotene accumulation in RNA interfered and control strains. (A) Shows the relative transcription levels of btwc-2a in RNA interfered and control strains at different times. The control strain, interfered strain 1 (interfererence 1), and interfered strain 2 (interfererence 2) were B. trispora strains transformed with pCambia1303-mU6-RNAidz, pCambia1303-mU6-btwc-2a-RNAi1, and pCambia1303-mU6-btwc-2a-RNAi2, respectively; (B) shows the accumulation of β-carotene in btwc-2a interfered (strain 1) and control strains at different times under different light conditions; (C, D) show the relative transcription levels of carB and carRA in btwc-2a interfered (strain 1) and control strains at different times, respectively. Dark denotes that strains were cultured in the darkness, while light represents that strains were exposed to blue light (1,000 lux) for 30 min and then moved into darkness. All strains were pre-cultured in the darkness for 3 days, at which time the transcription levels of these genes were used as controls. Three biological replicates were conducted for each experiment.