Cloning and activity of the crgA promoter from Blakeslea trispora

Abstract:

[Background] CrgA is a key negative regulator of carotenoid synthesis in Blakeslea trispora (Bt). [Objective] To clone and determine the activity of the crgA promoter from B. trispora, laying a foundation for deciphering the regulatory mechanism of CrgA expression. [Methods] According to the genome sequence in the integrated microbial genomes (IMG), we cloned a sequence of 2 000 bp upstream of the translation initiation site of crgA, identified the cis-regulatory elements, predicted the transcription initiation region, and then Analysis of crgA transcription level of B. trispora under different light time by RT-qPCR. Furthermore, we constructed four recombinant expression vectors p1303-procrgAF, F1, F2, and F3 driven by truncated sequences of crgA promoters with different lengths, and integrated them into the genome of B. trispora via Agrobacterium tumefaciens. Under dark and light conditions, the fluorescence signals were observed and the β-d-glucuronidase (GUS) activity was determined. [Results] The crgA promoter contained not only the basic elements TATA-box and CAAT-box but also multiple elements related to light response. Both CaMV35S and the constructed four mutant promoters could drive the expression of downstream genes in B. trispora. The GUS activity indicated that procrgAF3 had the strongest activity, and the truncated promoter had stronger activity than CaMV35S. The btcrgA promoter sequence has a light-responsive element at 1 499−989 bp that activates downstream gene expression. [Conclusion] The transcription of crgA in B. trispora presented the phenomena of light activation and light adaptation. The core region of the crgA promoter was identified as procrgAF3, and the cis-acting elements on the btcrgA promoter that responded to light regulation were located in the region of 1 499−989 bp.