| Astaxanthin is widely used in medical,food,cosmetics and aquaculture industries due to its strong antioxidant capacity.Haematococcus pluvialis（H.pluvialis）,as a best biological source for astaxanthin production,is facing the bottleneck of high cost and low yield.Proline（Pro）is an important molecule in regulating plant stress resistance,which can regulate cell osmotic pressure,maintain protein,membrane stability and scavenge reactive oxygen species（ROS）.However,the role of Pro in H.pluvialis has not been reported.In addition,as a plant growth regulator and antioxidant,melatonin（MT）has been proved to promote the synthesis of H.pluvialis under abiotic stress and putrescine（Put）,as a"second messenger"under stress,plays an important role in the regulation of plant growth and metabolism.Unsatisfactorily,the regulatory effect of putrescine on H.pluvialis induced by MT is not clear.In the study,H.pluvialis LUGU,a high astaxanthin producing strain,was used as the research object.Firstly,the ways of abiotic stress culturing H.pluvialis were optimized.Then,the effect of exogenous Pro on astaxanthin accumulation in H.pluvialis was explored,and the experimental results were verified by multiple algae species.Besides,the culture system of astaxanthin synthesis in H.pluvialis was constructed by abiotic stress coupled with chemical induction and the effects of put combined with MLT on Astaxanthin Accumulation under abiotic stress were studied.Furthermore,the relationship among putrescine（Put）,ROS andγ-aminobutyric acid（GABA）signaling molecules for the biosynthesis of astaxanthin was elucidated,and the regulatory mechanism of endogenous Put on Astaxanthin synthesis in H.pluvialis under abiotic stress was revealed,which provided a theoretical basis for promoting the commercial production of H.pluvialis.The results are as follows.（1）Under light intensity of 250μmol m-2s-1,1 g L-1Na Cl,at the initial biomass density of 3.5×105 cells m L-1 and ND conditions,the highest astaxanthin content was24.02 mg g-1.Further,the concentration of Pro was optimized under multiple abiotic stresses.The results show that the biomass,astaxanthin content and productivity of H.pluvialis LUGU are 1.43 g L-1,29.43 mg g-1 and 3.45 mg L-1d-1 after applicting 100μM Pro,which are increasing1.19-,1.24-and 1.98-fold,respectively.The application of Pro upregulates the lipid content,transcription levels of carotenogenesis and lipogenesis genes,accelerates the synthesis of chlorophyll and protein and reduces the carbohydrate content.Furthermore,the level of antioxidant enzyme gene,Put and MT are up-regulated,but reduces the levels of MDA and ROS.Therefore,it is inferred that exogenous Pro may reduce the oxidative damage in algal cells by increasing the levels of chlorophyll and protein,activating the activity of antioxidant enzymes and scavenging the burst of ROS.On the other hand,Pro regulates the levels of endogenous Pro,Put and MT,which can promote the efficient accumulation of astaxanthin in Haematococcus pluvialis.（2）These results suggest that MT and Put may be involved in the signal regulation of astaxanthin accumulation in H.pluvialis.Hence,an abiotic stress coupled with chemical induction（MT combined with Put）was constructed to promote the efficient astaxanthin synthesis of H.pluvialis.The results that under the conditions of 15μM MT coupling with 100 n M Put,the highest axtaxanthin and lipid content are 3.64%and 55.84%,which incrases 1.17-and1.17-fold than control group.Moreover,the combined application of MT and put also promotes the expression levels of carotenogenesis,lipogenesis genes,as well as sod and cat.The level of ROS is decreased and the levels of GABA and Put are increased.（3）In addition,the mechanism of endogenous Put was verified.The results show that under MT combined with Put conditions,the contents of astaxanthin and lipid in MT combined with Put are increased than MT treatment alone,while under the conditions of MT combined with DFMO（Put synthesis inhibitor）,the synthesis of astaxanthin and lipid are inhibited.MT combined with Put further increase the expression levels of carotenogenesis and lipogenesis genes,reduce the level of ROS,enhance the levels of endogenous GABA and Put and up-regulate the expression levels of sod,cat and odc,and vice versa.Consequently,under MT treatment,Put could improve the resistance and astaxanthin accumulation of microalgae by regulating Put,GABA and ROS.This study constructed a system to promote the co-accumulation of astaxanthin and lipid,elaborated the relationship between MT,Put,signal molecules and astaxanthin synthesis of H.pluvialis,which provided a theoretical basis for efficient astaxanthin accumulation of H.pluvialis. |
