Protective Effects Of Melatonin On Chlamydomonas Reinhardtii Under Salt Stress

Melatonin(MT;N-acetyl-5-methoxytryptamine)is a natural derivative of tryptophan and is a central biomolecule synthesized in vivo by almost all organisms including animals and plants.Melatonin is a naturally occurring heterocyclic ring compound that reacts with hydroxy(-OH)and alkyl peroxy radicals(ROO-).Melatonin is involved in many cellular actions as an antioxidant and is an excellent free radical scavenger both in vitro and in vivo.Melatonin participates in the protection of plants against various abiotic stresses.Chlamydomonas reinhardtii is a kind of unicellular eukaryotic green algae,which has a fast growth rate,complete genome sequencing and simple molecular genetics,and has become a popular microalgae model organism.Chlamydomonas is used for research on plant functional genes and secondary metabolites.However,studies on melatonin in microalgae are still relatively weak.In this study,Chlamydomonas reinhardtii was used as a receptor to perform the physiological effects of exogenous melatonin and overexpression of endogenous melatonin on Chlamydomonas reinhardtii in response to salt stress.The aim is to investigate the protective effect of melatonin on the growth of microalgae in salt stress,and to provide reference for the functional study of melatonin in microalgae.On the one hand,the paper studied the response of Chlamydomonas reinhardtii to salt stress under different concentrations of exogenous melatonin.Algae growth,morphological observation,membrane lipid peroxidation,antioxidative enzyme activity and intracellular melatonin concentration were used as measurement indexes.On the other hand,by constructing a Chlamydomonas expression vector p DBle-AANAT which harboring one of the melatonin syntheses arylalkaylamine N-acetyltransferase,AANAT.AANAT was introduced into this model microalgae genome by glass bead transformation using Chlamydomonas reinhardtii as receptor.The AANAT transgenic algae strain was identified by resistance screening,culture and molecular biology methods.The changes of the content of melatonin in the transformed algal strains and non-transformed algal strains were detected,and the antioxidant capacity of the algal strain overexpressing AANAT under salt stress was analyzed.The main findings are as follows:(1)The protective effects of exogenous melatonin on Chlamydomonas reinhardtii under salt stress:(1)The salt stress treatment concentration was 100 m M Na Cl.The results showed that 0.1?M and 1?M MT promoted the growth of Chlamydomonas cells,but the 10?M and 100?M MT exhibited negative effects on growth;(2)1?M MT could maintain intact cell morphology or cellular aggregation of Chlamydomonas in an unfavorable salt environment.(3)The presence of 0.1 ?M,1 ?M and 10 ?M MT led to increased activity of antioxidants SOD,CAT,and POD which can help cells eliminate toxic substances,and reduced the level of MDA(the peroxidation product of the cell membrane),which can help cells reduce oxidative damage.(2)The specific nuclear expression vector p DBle-AANAT containing AANAT was constructed: The plasmid PUCK-AANAT and p DBle were digested with Nde I and Eco R I,and then ligated with T4-DNA ligase to obtain the recombinant plasmid p DBle-AANAT.Finally,after PCR,double enzyme digestion confirmed that the vector was successfully constructed.(3)Identification of transforming Chlamydomonas reinhardtii strains to obtain positive transformed algal strains: The recombinant plasmid p DBle-AANAT was introduced into the nuclear genome of Chlamydomonas reinhardtii(The algal strain was the CC-503 cell wall deletion strain)by glass bead transformation.The transformed algal strains were screened and cultured with Ble resistance,and the DNA and RNA of the transformed Chlamydomonas were extracted and subjected to PCR for Ble and q RT-PCR for AANAT gene detection.Finally,a positive transformed C.reinhardtii strain Tn was identified.(n is different transgenic line).(4)Physiological performance of AANAT transformed algae strains under salt stress:(1)The concentration of melatonin in the cells of different AANAT transformed algae strains was determined and found that the concentrations of melatonin in the transformed algal strains T1,T2,T4,T5,T6,and T8 were significantly(P < 0.01)higher than those of untransformed algal strains.(2)Subsequently,the relevant indicators of the antioxidant system in different strains of algae were measured at a concentration of 100 m M Na Cl stress.The results showed that compared with untransformed algal strains,the activities of the three antioxidant enzymes catalase(CAT),superoxide dismutase(SOD)and peroxidase(POD)in the transformed algal strains T1,T2,T4,T5,T6,and T8 cells were all significantly higher at the P < 0.01 level.At the same time,the content of malondialdehyde(MDA)in the cells of the transformed algal strains was significantly reduced at the P < 0.01 level.