| β-carotene is widely used in food,feed,medicine and other fields,and the filamentous fungus Blakeslea trispora is the most commonly used β-carotene industrial production strain,and the accumulation of β-carotene is affected by light.But the biosynthesis mechanism ofβ-carotene,especially the light induction mechanism is not clear yet,which restricts the use of light to regulate the high-level synthesis of carotenoids.In response to the above-mentioned problems,this thesis takes B.trispora and its photoreceptors as the research target,and carries out the following researches.(1)Lay the foundation of this research from the perspective of media and genome extraction method optimization.The effects of 12 fungal culture media on the growth,sporulation ability and carotenoid synthesis of B.trispora mycelium were compared.The results showed that in the early growth period(2 d),the mycelium cultured on the modified Martin’s medium had the strongest growth rate and sporulation ability;in the late growth period(6 d),the growth and sporulation ability of mycelium cultured on the BAY medium are the best;the mycelium cultured on 5° wort medium has the strongest carotenoid-producing ability.In addition,a method for rapidly extracting the genome of B.trispora was constructed.Take a small amount of lawn in 40-50 m M Na OH solution and let it stand for 10 min,and take the supernatant as the genome source for subsequent PCR amplification and other experiments.The method has the advantages of simple operation,high concentration of the extracted genome,time-saving,labor-saving,and economical.The method can also be used to operate a large number of samples,which greatly improves the research efficiency.(2)The effects of different pulsed illumination modes on the synthesis of carotenoids in B.trispora were investigated.First,an efficient extraction method for β-carotene was established.Select the non-polar solvent ethyl acetate as the extraction agent,small volume,low temperature,short time,multiple extractions,and relatively high β-carotene content can be obtained.The light test results show that both light pulse time and light quality will affect the synthesis of carotenoids.Culture the mycelium in the dark for 3 d,irradiate it with blue light for 1 h,and then turn to dark conditions to continue culturing for 3 d.Under this condition,the accumulation of β-carotene is the highest and the mycelium grew well;in different pulsed light modes(dark culture for 3 d,light for 1 h+culture in darkness for 3 d;light for 1 h+ culture in darkness for 11 h,repeated 6 times;light for 1 h+ culture in darkness for 23 h,repeated 3 times),there was no significant difference in the effect on the accumulation of B.trispora β-carotene.However,with the prolongation of the total light time,the accumulation of β-carotene slightly increased;during the course of 1 h of light and within12 h after the light was switched to the dark,the accumulation of β-carotene of the bacteria was lower than that of continuous darkness group,it shows that after the start of light,the cell rapidly consumed intracellular β-carotene,and the light-induced carotenoid biosynthesis was lower than the carotenoid consumption when it resists oxygen stress.Light has a positive regulatory effect on the enzyme activities of phytoene synthase,phytoene dehydrogenase and lycopene cyclase.(3)Preliminary excavation was carried out on some regulatory elements that affect the synthesis of carotenoids by light.Firstly,the sequence of the photoreceptor component BTWC-2 of B.trispora was determined and analyzed by bioinformatics.The results showed that it has a PAS domain and an atypical zinc finger DNA binding domain.Then the sequence of the carotenoid structural gene hmg R was determined.Through amino acid sequence alignment and domain analysis,it was confirmed that it contains the conserved domain of the hmg R superfamily and has high homology with the HMGR protein of other filamentous fungi.The hmg R promoter sequence was analyzed,combined with website prediction and structure analysis,a total of 20 sequences of carotenoid structural genes hmg R,car B and car RA upstream were selected to synthesize probes for gel migration experiments,and 7 possible light responses were screened out.Original document,and proved that in vitro,hmg R,car B and car RA may all be under the transcriptional regulation of the three types of WC-1,WC-2may also have transcriptional regulation of hmg R,and WC-1/2 has the effect on the above three genes.Transcription regulation may be the key reason for the increase of the enzyme activities of phytoene synthase,phytoene dehydrogenase and lycopene cyclase 1-6 h after light,and the increase of carotene accumulation after turned to dark for 2-3 d after cultivation. |
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