| As an environmentally friendly energy source,biodiesel has received more and more attention.Microalgae oil is one of the sources of biodiesel,its production is being studied by many people.And beta-cryptoxanthin is a kind of pigments with great significance to hμman health,which the research on beta-cryptoxanthin is also very extensive.In this paper,D.tertiolecta was used as the research object to study the effects of piperonyl butoxide and imidazole on the accμmulation of oil andβ-cryptoxanthin,respectively,and the combination of different light conditions.To increase the lipid andβ-cryptoxanthin accμmulation of D.tertiolecta,the effect of piperonyl butoxide on the transcriptional level of MFPα,a lipid metabolism-related gene of D.tertiolecta,and the effects of the transcriptional level of ZEP and BHY,the key to the metabolism of carbotenoids(Carotenoid)by D.tertiolecta.Experimental results related to piperonyl butoxide showed that the light intensity of 8000 lux which is most conducive to oil accμmulation.Under these conditions,piperonyl butoxide at150 ppm(ppm=1 mg/L)can increase the final accμmulation of D.tertiolecta oil and the oil content per cell up to 21.79%and 76.42%respectively.Laser confocal microscopy cytogram of D.tertiolecta stained by Bodipy 505/515 showed that the fluorescence of lipids in D.tertiolecta cells treated with piperonyl butoxide increased,which also confirmed that the addition of piperonyl butoxide was beneficial to the accμmulation of lipids in D.tertiolecta cells.The transcription level of DtMFPαin the lipid metabolism pathway of D.tertiolecta was increased by nearly 31.52 times in the presence of piperonyl butoxide at 150 ppm.It can be inferred that the accμmulation of oil in D.tertiolecta is probably related to the increased expression of DtMFPαin D.tertiolecta under the action of piperonyl butoxide.The standard curve ofβ-cryptoxanthin content measured by HPLC was y=0.0069x-0.1292,R~2=0.9999.The correlation coefficients showed that the determination of beta-cryptoflavin by HPLC was reasonable.In the experiment of optimizing the concentration of imidazole,imidazole at 0-1000 ppm was combined with different illμmination conditions of 6000 Lux,8000 Lux and 10000 Lux,the optimμm conditions for accμmulation of beta-cryptoxanthin in D.tertiolecta can be obtained as follows:8000 lux light intensity,96 hours under the condition of adding imidazole at 500 ppm.And the final peak area of beta-cryptoxanthin in D.tertiolecta was 57.85 mAU*s,and the accμmulation of beta-cryptoxanthin in D.tertiolecta was 0.05 mg/L.With the addition of imidazole,the expression of DtZEP and DtBHY in Carotenoid synthesis pathway in D.tertiolecta was significantly increased,with the highest expression of DtZEP and DtBHY at 750 ppm.DtZEP is the downstream synthesis gene of beta-cryptoflavin and DtBHY is the upstream synthesis gene of beta-cryptoflavin.Theoretically,the down-regulation of DtZEP gene expression and the up-regulation of DtBHY gene expression are more conducive to the accμmulation of beta-cryptoflavin.DtZEP and DtBHY play an important role in many nodes of Carotenoid synthesis pathway.Therefore,it can be speculated that the accμmulation of beta-cryptoflavin may be related to the effect of imidazole on multiple nodes and other key enzyme genes of Carotenoid synthesis pathway in D.tertiolecta. |
