Proteomics Of Two Species Of Intertidall Macroalgae Under The Stress Of Heavy Metal Copper

With the development of agriculture and modern industry,the heavy metal emissions in the environment are increasing.Heavy metal pollutants enter the water body through participating in the complex biogeochemical cycle of the water body,leading to the increasingly severe living environment faced by marine plants.The intertidal zone,as the boundary between the ocean and the land,has its special geographical features,and the microenvironment changes greatly.The intertidal macroalgae inhabiting on the rocks and reefs in the intertidal zone or in the rock marshes are also facing the increasing stress of heavy metals.At present,more and more attention has been paid to the study of marine organisms by proteomics.2-DE technology can be used to compare samples under different environmental stresses,determine specific proteins related to treatment effects,and comprehensively understand the effects of stress on organisms.However,up to now,there are few systematic reports on the specific changes of protein expression of macroalgae in intertidal zone under different concentrations of copper.Therefore,two representative intertidal macroalgae,Corallina pilulifera?Rhodophyta?and Sargassum thunbergii?Phaeophyta?,were selected as materials in this paper.The two-dimensional electrophoresis system of algae was optimized by proteomic methods and techniques,and the differential proteins of C.pilulifera and S.thunbergii under heavy metal stress were systematically optimized by two-dimensional electrophoresis Analysis.By analyzing the changes of protein types and expression abundance of algae under Cu²⁺treatment and analyzing the correlation between the gradient concentration of heavy metal Cu²⁺and protein expression,it can further lay a foundation for the study on the interaction between the stress response of coralline alga against copper stress and the environmental conditions under external stress.The research results of this paper are as follows:?1?A series of optimization of total protein extraction and 2-DE electrophoresis conditions of C.pilulifera were carried out,One is to optimize the extraction conditions of C.pilulifera proteins.The optimal method is to extract C.pilulifera proteins by phenol extraction.The protein separation is good,the map background is clearer,and there are no obvious horizontal and vertical stripes.The second is the optimization of the pH condition of the adhesive strip.When the pH of the adhesive strip is 4-7,the protein points are well separated on the gel,the protein points are evenly distributed,the overlap between protein points is less and the boundaries between protein points are obvious,so more protein points can be separated and the electrophoretic resolution can be improved.Thirdly,the concentration condition of the separation gel was optimized.The separation gel concentration of 12.5%was more suitable for the separation of the total protein of coralline algae by two-dimensional electrophoresis.And a 2-DE system suitable for total protein of C.pilulifera was established,which laid an effective foundation for the proteome study of C.pilulifera and provided an important reference for other macroalgae proteomics studies.?2?After the establishment of the two-dimensional electrophoresis system,the differential proteins of C.pilulifera under heavy metal copper ion stress were analyzed by two-dimensional electrophoresis.Cu²⁺could have a significant effect on the protein expression of C.pilulifera.The protein concentration under the treatment of 0.5 mg/L Cu²⁺reached a significant level of difference with the control,while under the treatment of the highest concentration of 2.0 mg/L Cu²⁺,the protein concentration significantly decreased compared with the control,showing the effect of"low promotion and high inhibition".The most significant difference was found between the 2D histone map of0.5mg/L Cu²⁺treatment and that of the control group.Nine of the 80 differentially expressed proteins were up-regulated and 13 were down-regulated.Mass spectrometry analysis identified four positive results,respectively,ABC transporters,SOD,PS?12kD external protein,conservative assumption.These proteins may play an important role in maintaining osmotic pressure balance,antioxidant enzyme activity and oxygen release rate.?3?Heavy metal copper ion has a significant effect on the content of protein in the body of S.thunbergii.At the concentration of 0.5mg/L,the quantity,species and expression of protein in the body of S.thunbergii have obvious changes.Compared with the CK group and the 0.5 mg/L group,151 proteins in the S.thunbergii showed significant changes.Compared with the CK group,the number of protein spots in the 0.5 mg/L group decreased by 104,36 were up-regulated and 11 were down regulated.From the analysis of the experimental results,it can be seen that the heavy metal copper ion will cause more obvious damage to the S.thunbergii at a lower concentration,which is irreversible.Under the stress of copper ion,the protein expression in the S.thunbergii is significantly inhibited and the systematic resistance of the S.thunbergii to copper ion is relatively poor.