Study On The Priming Effect And Mechanism Of Linolenic Acid On Prototheca Platensis

Red tides are marine ecological anomalies.In recent years,with the rapid development of China's agriculture and industry,eutrophication of water,the frequency of red tide increased,especially the frequent occurrence of toxic and harmful red tide,red tides threaten human health as well as the ecological environment problems,and causing significant losses to the economy.There are three methods of physical,chemical and biological methods to control red tide,in which use of allelopathic substances produced of macroalgae on inhibitting the outbreak of harmful algae has unique advantages,such as sustainable,environmentally friendly and so on.We selected one dominant red tide species Prorocentrum donghaiense as the research subject,to study the allelopathic effects and cell apoptosis mechanism of?-linolenic acid on P.donghaiense.In this study,the mechanism of inhibition of algae was revealed from a new perspective,providing scientific basis for prevention and control of red tide caused by P.donghaiense,so it has great theoretical significance.The specific results are as follows:1. Effects of?-linolenic acid on the growth and physiological and biochemical level of P.donghaienseLow concentration of linolenic acid could promote the cell growth of P.donghaiense?negativeinhibition?.With the increase of linolenic acid concentration,the growth of P.donghaiense decreased and linolenic acid inhibited its growth.The soluble protein content of algal cells also increased first and then decreased.Under the treatment of?-linolenic acid,the photosynthetic pigment content of P.donghaiense was reduced,the photosynthetic efficiency of algal cells was decreased,and the activity of nitrate reductase was increased first and then descended.2.Research on ROS and Ca²⁺signal pathway of?-linolenic acid to P.donghaienseThe activity of CAT,SOD,GPx,GST,GR and the contents of GSH/GSSG,As A/DHA in P.donghaiense cells treated with different concentrations of linolenic acid showed the trend of rise in the first stage,and then decreased.The total ROS level increased,and the content of MDA increased sharply.The results showed that ROS signal pathway was involved in the process of allelopathic substances linolenic acid effecting on P.donghaiense.The activity of Ca²⁺-ATPase of P.donghaiense after treatment with different concentrations of linolenic acid showed significant changes,the results showed that Ca²⁺signal pathway was involved in the process of allelopathic substances linolenic acid effecting on P.donghaiense.Ca Cl₂ could increase the activity of GPx of antioxidant enzyme,while adding extracellular calcium chelator EGTA,plasma calcium channel blocker La Cl₃,and intracellular calcium chelator BAPTA,can significantly inhibit the activity of GPx.It is postulated that Ca²⁺participated in the oxidative stress process of P.donghaiense after linolenic acid treatment.Moreover,the increase of Ca²⁺in the cytoplasm mainly comes from the release of extracellular Ca²⁺influx and intracellular Ca²⁺release.After adding NAC,the activity of calcium pump was inhibited significantly.It was hypothesized that ROS could be involved in the Ca²⁺signaling pathway of P.donghaiense.3.Research on the mechanism of cell apoptosis in P.donghaiense under?-linolenic acid exposureThe surface structure of P.donghaiense was observed by scanning electron microscopy.The results showed:cell shape changes significantly,and the cells appeared to be ruptured;The cells were not clear and the floc appeared on the cell surface.The flocculation increased with the increase of linolenic acid concentration.The cell ultrastructure was observed by transmission electron microscopy.The results showed:the plasmalemma separation of P.donghaiense was observed in the linolenic acid treated group and cell volume becomes smaller;Cell wall,cell membrane,and vacuole membrane are dissolved lead to fracture;Chloroplast membrane boundaries blurred,and the basic granular lamellar structure loose or even disappear;Most cells appear disintegrated.The phosphorylated serine,which appeared on the surface of the cell membrane,was detected by Annexin V labeled with green fluorescence.The results showed that most of the cells entered the late stage of apoptosis after treated with?-linolenic acid.Under ALA exposure P.donghaiense showed some changes,such as MMP decreased,the release of Cyt-C increased,the content of Bcl-2 increased,and the activity of Caspase-9,3increased.These indicators confirm that allelopathic sbustances could induce cell apoptosis by the mitochondrial pathway.