| In recent years,with the development of industrialization and globalization of human society,water environmental degradation has become the restriction factor of human existence and development.Red tides are marine ecological anomalies.The world freshwater and coastal areas of nutritionalization lead to the frequent occurrence of red tide,which poses a serious threat to the sustainable development of aquaculture industry,human health and marine ecological security in coastal areas.At present,there are physical methods,chemical methods and biological methods to control red tide.Among them,using of allelopathic substances produced by macroalgae on inhibitting the outbreak of harmful red tide has good effect,and will not cause secondary pollution to environment and the damage of ecological system,are increasingly brought to the attention.This research adopts the experimental method of ecology,we selected one dominant red tide species Karenia mikimotoi as the research object,to study the allelopathic effects and cell apoptosis mechanism of K.mikimotoi under the stress of linoleic acid.This study provides a theoretical and practical basis for the development and utilization of algal bloom and new type of algae.The main results are as follows:1.Effects of LA on growth and photosynthesis of K.mikimotoiThe microalgal K.mikimotoi continues to growing under the low concentration(<500 ?g/L),but with the high concentration of linoleic acid(>500?g/L),the cell growth is restrained,appeared the phenomenon of the cell growth was promoted by low concentrations and inhibited by high concentrations.Flow cytometry detection data found that high concentration of linoleic acid resulted in the cell cycle was arrested at G2 phase.The soluble protein content of algal cells elevated first and then reduced.Under the treatment of linoleic acid,the content of soluble protein and photosynthetic pigment of K.mikimotoi was increased first and then was reduced,the optimal/maximal PS?efficiency and the performance index on absorption basis of algal cells was decreased.This indicates that the photosynthetic efficiency of algal cells is impaired.2.The oxidative stress response of K.mikimotoi under the stress of linoleic acidReactive oxygen species(ROS)levels were significantly increased under the stress of LA,and the equilibrium of REDOX state was broken.At the same time,intracellular antioxidant enzymes SOD and CAT activity showed low concentrations promote inhibition of high concentration trend.The content of malondialdehyde(MDA)elevated with the increase of linoleic acid concentration,indicating that the lipid peroxidation of algal cell membrane was increased.Moreover,after the addition of antioxidant n-acetyl-l-cysteine(NAC),the flow cytometry results showed that the intracellular ROS level was lower than that of linoleic acid stress group.3.Research on the mechanisms of cell apoptosis in K.mikimotoi under linoleic acid exposureThe surface structure of K.mikimotoi was observed by scanning electron microscopy.Results showed: cell shape changes significantly,and the cells appeared to be ruptured,there are much attachment appeared on the cell surface.The activity of caspase-3 enzyme and the degree of DNA fragmentation were increased in a dose-dependent manner.The fluorescent probe FITC labeled with green fluorescence was used to detect the Annexin V which appeared on the surface of the cell membrane.Results show that different stages of apoptosis were found in the cells of K.mikimotoi with linoleic acid treatment,and the higher the concentration,the greater the apoptosis degree.Under the stress of linoleic acid,the apoptosis of algal cells decreased after adding Caspase-3 specific inhibitor Ac-DEVD-CHO.The addition of antioxidant NAC can inhibit the intracellular activity of Caspase-3 enzyme,and alleviate the apoptosis of algal cells.The above results can be inferred that Caspase-mediated apoptosis of K.mikimotoi caused by oxidative stress under the stress of linoleic acid is an important apoptosis way. |
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