Identification And Preliminary Functional Analysis Of Photoreceptors In Blakeslea Trispora

As a kind of pigment,carotenoids have highly effective anti-oxidation,anti-aging and anticancer functions,and are widely used in the fields of medicine,food and cosmetics.Blakeslea trispora is an industrial strain that synthesizes ?-carotene in large quantities and its synthesis process is regulated by light.However,there have been few reports on the physiological process and molecular mechanism of photoregulation of the synthesis of carotenoids in B.trispora.This study starts from photoreceptors in B.trispora based on the present researches,including separation and identification of three photoreceptors in B.trispora,called Blakeslea trispora White Collar-1(BTWC-1).And a systematic study was carried out on the characteristics and functions of photoreceptor proteins in order to lay a foundation for the physiology and molecular biology of light regulation in B.trispora.The specific contents are as followed:1)Cloning,bioinformatics analysis and heterologous expression of Blakeslea trispora photoreceptor.Three photoreceptor proteins were named BTWC-1A,BTWC-1B and BTWC-1C.Bioinformatics analyses of these genes and their protein sequences revealed that their functional domains(Light-Oxygen-Volt(LOV)domain and zinc finger structure)have significant homology to other fungal blue-light regulator proteins from Mucor circinelloides and Neurospora crassa.The photoreceptor proteins were synthesized by heterologous expression in Escherichia coli.2)Analysis in vitro of photosensitization of BTWC-1.The chromogenic groups FAD and FMN were detected to accompany BTWC-1 proteins by using high performance liquid chromatography(HPLC)and fluorescence spectrometry,demonstrating that the proteins may be photosensitive.The absorbance change of the purified BTWC-1 proteins in dark and light indicated that they were light responsive and underwent a characteristic photocycle by light induction.Site-directed mutagenesis of the cysteine residual(Cys)in BTWC-1 did not affect the normal expression of the protein in E.coli,but led to the loss of photocycle response,indicating that Cys is flavin-binding domain for photon detection.3)Analysis in vivo of BTWC-1 function based on photoreceptor knockout strain of Mucor circinelloides.The functions of BTWC-1 proteins were then analyzed by complementing btwc-1a,btwc-1b and btwc-1c into their counterpart knockout strains of M.circinelloides for each mcwc-1 gene.Transformation of the btwc-1a-complement into mcwc-1a knockout strains restored the positive phototropism,while the addition of btwc-1c-complement remedied the deficiency of carotene biosynthesis in the mcwc-1c knockout strains under illumination.These results indicated that btwc-1a and btwc-1c is involved in phototropism and light-inducible carotenogenesis.