Study On The Killing Effect And Apoptosis Mechanism Of Linoleic Acid To

Since the 1960 s, the phenomenon of harmful algal blooms(HABs) began to spread around the world, it has become one of the world’s serious marine disasters. The frequent occurrence of HABs in coastal areas of the world, resulting in a significant adverse impact on public health, the marine environment, aquaculture and natural resources. In the face of the complexity of the HABs problem and the severity of the red tide disaster, actively study and take practical and effective measures for prevention of HABs is imminent. Numerous studies show that unsaturated fatty acids secreted from macroalgae on HABs microalgae produce allelopathic effect is a kind of environmentally friendly, sustainable and strong biological control methods. In this experimental thesis, we selected two representative advantages HAB species Heterosigma akashiwo and Prorocentrum donghaiense as the research subjects, and selected unsaturated fatty acids linoleic acid as allelochemicals. Studies the influence of linoleic acid on Heterosigma akashiwo and Prorocentrum donghaiense allelopathic effect and effects on apoptosis mechanism.The purpose of this research lies in offer practical scientific basis for using macroalgae effectively control of harmful algal blooms implementation, which has important theoretical significance and application prospects. The results are as follows:1. The effect of different concentrations of linoleic acid on Heterosigma akashiwo and Prorocentrum donghaiense with 48 hours, preliminary study on the inhibitory effect of linoleic acid on Heterosigma akashiwo and Prorocentrum donghaiense. Result shows that with the increase of the concentration gradient of linoleic acid, the growth of microalgae showed a trend of first increasing and then decreasing. Meanwhile, the inhibition rate of the two species of microalgae showed a trend of first decreased and then increased. When the concentration of linoleic acid was 900μg/L, the inhibition rate of Heterosigma akashiwo was 97.3%, and the inhibition rate of Prorocentrum donghaiense was 91.8%. The inhibition effect of linoleic acid on two kinds of microalgae was significant(P<0.05).2. Through effect of different concentrations of linoleic acid on Heterosigma akashiwo and Prorocentrum donghaiense, detecting the antioxidant system, the level of reactive oxygen species and the level of membrane lipid peroxidation. Result shows that with the increase of the concentration of linoleic acid, intracellular antioxidant enzymes SOD, CAT, POD activity and antioxidation non enzyme VC and GSH content showed low concentrations promote inhibition of high concentration trend. Total ROS and O2-· level presented the trend of first increases and then decreased. The overall trend of H2O2 and ·OH level showed upward. The content of MDAincreased continuously with the increase of linoleic acid concentration.3. Under the influence of linoleic acid, from cell morphology, biochemical and molecular biological level, by trypan blue staining, transmission electron microscopy, flow cytometry and fluorescence methods to detect apoptotic mechanism. The results are as follows.(1) With linoleic acid concentration increased, and the number of Heterosigma akashiwo and Prorocentrum donghaiense living cells showed a trend of first rising and then falling. When linoleic acid reaches maximum concentration 900μg/L, the number of Heterosigma akashiwo living cells was reduced by 85.9%, the number of Prorocentrum donghaiense living cells decreased 85.9%. Compared with the control group, the difference was significant(P<0.05).(2) Under the influence of linoleic acid, the ultrastructure of the cells showed that the cell shape of Heterosigma akashiwo is small and irregular, cytoplasmic shrinkage, part of the cell membrane, mitochondrial membrane, chloroplast membrane is not clear even have disappeared,only fragments. Prorocentrum donghaiense cells showed plasmolysis, cell wall and vacuole membrane rupture occurs, the cell appears empty, significant expansion of the chloroplast, grana appear loose or even disappear.(3) The fluorescent probe FITC labeled with green fluorescence was used to detect the V Annexin which appeared on the surface of the cell membrane. Result shows that different stages of apoptosis were found in the cells of Heterosigma akashiwo and Prorocentrum donghaiense which linoleic acid treatment.(4) Under the influence of linoleic acid, by JC-1 fluorescent probes in cells transformed from the red fluorescence to green fluorescence showed Heterosigma akashiwo and Prorocentrum donghaiense mitochondrial membrane potentia decline. The use of a microplate reader tests showed Caspase-3,9 activity and Cyt-C release significantly increased, Bcl-2anti-apoptotic protein expression was reduced.