Mechanisms Of Salinity Stress Coupling GABA Regulating Haematococcus Pluvialis Growth And Astaxanthin Synthesis

Astaxanthin is a super natural antioxidant.It is widely used in industries such as food,aquaculture,cosmetics,medical care and pharmaceuticals.It also has a high market value in the global economy.Haematococcus pluvialis（H.pluvialis）is the best source for the synthesis of natural astaxanthin,but it faces the challenge of high cost and low yield.Abiotic stress coupled with chemical induction and two-stage culture strategy are effective ways to promote the growth and astaxanthin accumulation in algae.Gamma-aminobutyric acid（GABA）is a non-protein amino acid,which is in the GABA shunt of the tricarboxylic acid cycle.It has an important physiological role in animals,bacteria and plants.However,the GABA signal transduction pathway in microalgae cells and its mechanism of action on the synthesis of secondary metabolites are not yet clear.In this study,H.pluvialis LUGU was used as the research object to study the mechanism of different concentrations of NaCl on the growth and metabolism of algae through two-stage culture.Meantime,the culture system of abiotic stress coupled with GABA for high astaxanthin productivity was constructed.The regulation of the signal molecule reactive oxygen species（ROS）and endogenous GABA on key genes in the pathway of astaxanthin and lipid synthesis under salinity stress and GABA induction was explored.The mechanism of astaxanthin synthesis induced by GABA was clarified.The internal connection between GABA and ROS signals in microalgae cells was explored.And the key signal transduction pathways were analyzed.The study provided a scientific basis and target for genetically engineering microalgae to increase the production of astaxanthin.It had certain theoretical and practical significance for the research on the efficient synthesis of astaxanthin by microalgae.The specific research results are as follows:（1）Using a two-stage culture method to explore the effect of sodium chloride（NaCl）on the growth and accumulation of astaxanthin in H.pluvialis.In the growth stage,12.5 mg L^-1NaCl significantly shortened the culture cycle of H.pluvialis,and the biomass reached 2.36 g L^-1on the 8th day of culture,which was 1.28 times that of no NaCl.Meantime,the content of chlorophyll and carbohydrates in algae cells increased,and the Fv/Fm and specific growth rate also increased significantly.In addition,the expression of growth-related genes rbc S,rbc L and nr were up-regulated,which were 2.78,2.18 and 2.59 times that of the control group,respectively.The results showed that low concentration of NaCl can achieve high yield of H.pluvialis biomass.（2）In the induction stage,the effects of NaCl on the growth and accumulation of metabolites of H.pluvialis under high-light stress were investigated.The results showed that the biomass,astaxanthin and lipid contents of 2 g L^-1NaCl group reached the highest,which were 1.53 g L^-1,25.92 mg g^-1,and 52.56%,respectively,compared with the control group increased significantly by 18%,112.81%,and 17.24%,respectively.Meanwhile,carbohydrate and protein levels in algae cells decreased,GABA content increased,Ca²⁺level and cam expression increased,and the decrease of ROS level was accompanied by the up-regulation of antioxidant enzyme genes（sod,cat）expression.In addition,the expression of genes related to astaxanthin and lipid synthesis was up-regulated.The results showed that high concentration of NaCl promoted the growth and accumulation of astaxanthin relating to the levels of GABA,Ca²⁺and ROS in H.pluvialis.（3）This study also established a culture system for salinity stress coupled with GABA to promote efficient accumulation of astaxanthin and lipids in H.pluvialis under high-light stress.The study found that the biomass,astaxanthin yield and lipid content of H.pluvialis under the induction of 0.25 mM GABA reached 1.65 g L^-1,3.86 mg L^-1d^-1,and 55.11%,respectively.Compared with the salinity control group,it increased by 8%,66%,and 12%,respectively.At the same time,carbohydrate and protein contents decreased,intracellular GABA content increased(19.21 mg g^-1),and gad expression increased.In addition,salinity stress coupled with GABA up-regulated microalgae growth,astaxanthin and lipid synthesis-related gene expression levels,and antioxidant enzyme gene expression levels,but reduced ROS levels.When the endogenous GABA decreased,the contents of astaxanthin,lipids and GABA in the algae cells decreased simultaneously,but the level of ROS increased.The above results indicated that salinity stress coupled with GABA can increase the accumulation of biomass,astaxanthin and lipids by regulating intracellular ROS and GABA signals,as well the expression of growth,astaxanthin and lipids synthesis-related genes.