Study On The Effect Of Chlamydomonas Sp. ICE-L Under Heat Stress

Antarctic ice algae are the microalgae living in extreme environments such as Antarctic sea ice and sea ice edge.The extreme polar environmental conditions created its special biological characteristics.Because of the global warming in recent years,the melting of sea ice is getting more and more seriously.The mechanism and ability to resist high temperature stress is one of the important factors for the survival of Antarctic ice algae.Therefore,the investigation of the responding mechanism of Antarctic ice algae under high temperatures has important theoretical significance and application value.In this paper,Chlamydomonas sp.ICE-L was studied as the object,the results are as follows:(1)Based on the differences in high-throughput sequencing of gene expression analysis,eight Hsp70 genes,three Hsp90 genes,one Hsp22 gene and two Hsf genes of ICE-L were screened.The results of q RT-PCR showed that,Ci Hsp70 B gene was the most sensitive to heat stress at 15 ?,and the relative expression of Ci Hsp70 B gene was 12.18 fold higher than the control after heating treatment for 3 h.Ci Hsp70E1,Ci Hsp70E2 and Ci Dna K were delayed in response to heat treatment,but the expression levels were up-regulated to 9.76,5.42 and 6.22 fold changes respectively.The maximum relative expression of Ci Hsp70 C and Ci Hsp70 D were 3.18 and 3.87 fold compared to the control respectively.The expression of Ci Hsp70 A and Ci Hsp70 G was not obvious.The expression level of Ci Hsp90 A,Ci Hsp70 B and Ci Hsp90 C were increased firstly with the relative change of 2.92,4.40 and 20.37 fold respectively after heating treatment for 48 h,and then decreased.Ci Hsp90 C was the most sensitive to high temperature stress.Ci Hsp22 responded to high temperatures quickly,and the expression level was significantly up-regulated and maintained at high level with a relative change of about 15 fold at 6 h.Ci Hsf2 had a lower expression level,but Ci Hsf1 could respond to high temperature quickly.The expression level of Ci Hsf1 sustained stability at a high level,and reached the maximum relative change of 10.88 fold after heating treatment for 120 h.(2)The Hsp70 s of Chlamydomonas sp.ICE-L were analyzed using NCBI,Ex PASy,Wo LF PSORT and MEGA 6.06.The results of bioinformatics analysis showed that the Ci Hsp70 sequences identified in ICE-L were 642 to 985 amino acids ranging in length.The functional domain prediction showed that all of them belonged to the Hsp70 superfamily.Subcellular location prediction showed that Ci Hsp70 A,Ci Hsp70 E and Ci Dna K were locatedin cytoplasm,Ci Hsp70 B and Ci Hsp70 D were located in chloroplasts,Ci Hsp70 C was located in mitochondria,and Ci Hsp70 G located in endoplasmic reticulum.Phylogenetic analysis showed that all of the Ci Hsp70 genes in ICE-L,except Ci Dna K gene,could find their homologous sequences in other green algae,especially have high homologous with C.reinhardtii and V.carteri f.nagariensis.The Ci Dna K in ICE-L has highly homologous to Dna K proteins from prokaryotes,especially has 100% homologous with the Dna K of Psychroflexus torquis,a psychrophilic bacterium isolated from Antarctic sea ice.To study the function of Ci Dna K deeply,the Ci Dna K gene was successfully transferred into C.reinhardtii 137 c.Testing its heat resistance showed that the transformed C.reinhardtii 137 c with Cidna K gene showed much higher rates of survival than the wild-type under high temperature conditions at 42?.Ci Dna K can enhance the heat resistance of the recombination strains.However,the protection became weakened with the prolongation of treatment time.(3)Detecting the changes of the antioxidase activity indicated that,POD activity changed most obviously.POD showed low sensitivity under heat stress at the beginning,but increased sharply from 72 h to 144 h,significantly increased about 70 fold than the basic level.The relative expression of Ci PEX5 was significantly up-regulated 18.11 fold than the control at 72 h.CAT activity increased significantly within 24 h under heat stress,and reached about 2 fold of the control until 72 h,then declined slowly.The relative expression level of Ci CAT(45467),one of the CAT coding genes,increased about 4.67 fold at 72 h after heat stress.SOD enzyme activity was inhibited under the heat treatment.The expression of four genes encoding SOD was also down-regulated.In addition,MDA content increased rapidly within 24 h indicating that the membrane system might be damaged at the beginning under heat stress,and then decreased and recovered to the level of cell tolerance until 144 h.In this study,the response of heat shock protein and antioxidant system in ICE-L under heat stress was studied for the first time,which provided a basis for further exploring the response mechanism of ICE-L to high temperature.