| The Arctic region is one of the most coldest region on earth.Recently,under the background of global warming,the polar ecosystem undergo increasing temperature fluctuations of the polar regions,diversity of species and biogeochemical cycles in polar aquatic systems attract increasing attention worldwide.Polar microalgae are the main primary producers and play a fundamental role in the polar ecosystem.Whether polar microalgae can multiply rapidly and adapt to the temperature variation of polar region affect its intrinsic niche in the ecosystem,and subsequently the structure of polar ecosystem community,and even the carbon sink of the ocean.Therefore,the adaptability of polar microalgae has been one of the hotspots of polar research.In addition,polar microalgae are extreme microbes that develop unique adaptive mechanism in extreme environments,for example producing anti-stress bioactive substances with special structure and high biological activity under extreme conditions,which have great potential for application.In this study,the Arctic Chlorella sp.(Chlorella-Arc)was used as the experiment species,which was isolated from the glacier melt water collected near the China's Yellow River Station in the Ny-Alesund area(78° 55'N;11° 56' E),Spitsbergen Islands(Svalbard,Norway).Physiological and biochemical changes of Chlorella-Arc was determined at different temperature treatments(3?,15?,24?).Then transcriptomics and proteomics analysis were performed to clarify the adaptive mechanism of Chlorella-Arc under adverse temperature from physiological,gene expression and protein levels.This will provide information for better understanding the adaptative mechanism of polar microbes to in response to the temperature variation of global warming.Our previous work found that Chlorella-Arc polysaccharide played an important role in resistance to temperature stress.The genes and proteins related to polysaccharide biosynthesis pathway were significantly up-regulated under high temperature stress.As one of polar microbial bioactive substances,polysaccharide has great potential for application in natural antioxidants.Thus,the extraction condition was optimized,and primary structure and antioxidant activity of Chlorella-Arc polysaccharide was determined in this thesis,to provide theoretical data for Arctic microalgal bioactive substances application.The main results are as follows:1.Chlorella-Arc was found able to survive at 3-24? and had good adaptive capacity to wide range of temperature.Under low temperature stress,Chlorella-Arc showed a lower content of pigments(indicated by chlorophyll a,chlorophyll b,carotenoid),lower photosynthetic activities(indicated by Fv/Fm,Fv/Fo,ETR,qP,Y ?)due to photoinhibition induced by low temperature.Chlorella-Arc reduced the size of LHC to alleviate the photoinhibition.The heat dissipation ability of Chlorella-Arc was enhanced at higher temperature.In addition,Chlorella-Arc had high content of intracellular soluble sugar(38.89±1.67 ?g/mL)and total soluble sugar(74.6 ± 8.45 ?g/mL)under high temperature stress.On the contray,Chlorella-Arc tended to accumulate lipids(35.4%)and proteins(9.3 mg/g)under low temperature.Chlorella-Arc had a special strategy to protect itself in response to high/low temperature stress.2.Transcriptome analysis on Chlorella-Arc was performed under three temperature treatments(3?,15?,24?)by Illumina Hiseq 4000.A total of 36,658 non redundant unigenes were obtained.The transcriptome data were verified by qRT-PCR,and the raw data had been uploaded to the SRA database of NCBI.We found that the numbers of up/down regulated genes in high temperature treatment were 3447/3109,and the numbers of up/down regulated genes in low temperature treatments were 1351/3109.3.Transcriptome analysis of Chlorella-Arc showed that low temperature induced up-regulation of genes encoding photosystem components,including PsbB(CP47),PsbC(CP43),PsbA(D1),PsaA and PsaB,resulting in the enhancement on the energy transfer from the light harvesting antennae to the photosystem reaction center.Genes involved in the biosynthesis of lutein,zeaxanthin and xanthophyll(lut5,lut1,zep)were predominantly upregulated under low temperature,indicating that the lutein cycle is actively involved in the mechanism of low temperature photoprotection.Combined with the change of biochemical components and transcriptome analysis,the allocation of carbon to different synthetic pathways of lipids and sugars under low and high temperature might represent an effective strategy to prevent overexcitation in response to high/low temperature stress.The C4-like pathway might also play an important role in the regulation of carbon allocation at different temperatures.4.Proteome analysis on Chlorella-Arc was performed under three temperature treatments(3 ?,15 ?,24 ?).A total of 2018 proteins were identified.938 and 760 significantly changed proteins were identified in,respectively,high and low temperature treatments.The correlation analysis on triscriptome and proteome was performed,and the number of differentially expressed proteins at both transcriptional and protein levels was 103,144 and 219 in the 3 groups HT vs CT,LT vs CT and LT vs HT.5.Different from transcriptional regulation,PS I and PS II-related proteins were not up-regulated significantly under low temperature stress,the core protein subunits of PS II(CP47),and photosynthetic carbon fixation key enzyme(Rubisco),were down-regulated,suggesting that Chlorella-Arc reduction of carbon fixation is the limiting factor of cell growth at low temperature,which was consistent with our previous physiological results.In addition,it was found that ATP synthase(?,?,b subunits)and key enzymes of TCA cycle(CS,IDH,SCLA)were up-regulated under both high and low temperature stress.Enhancing ATP synthesis efficiency will provide sufficient energy for repairing damages,biochemical components transport and growth of algae cells.6.Consistent with the transcriptional regulation,the protein levels of key enzymes involved in Glyconeogenesis pathway(PEPCK,FBP),starch synthesis pathway(SS,UGP2,glgC)and nucleoside synthesis pathway(GALE,PMM,UDGH)were up-regulated under high temperature stress,indicating that regulation of intracellular carbon to carbohydrates occurred at both transcriptional and protein level.Transcriptomics did not clarify why carbon allocation in Chlorella-Arc was diverted to lipids other than carbohydrate at low temperature.However,proteomics revealed that glycolysis pathway and PDH-dependent acetyl-CoA were enhanced,suggesting that intracellular carbohydrate might provide carbon skeleton to fatty acid synthesis via glycolysis.This mainly occurred at post-transcriptional regulation.Thus,glycolysis/gluconeogenesis was considered an important regulatory pathway that directs carbon flow to the synthesis of carbohydrate/fatty acid.Related proteins of ribosome(EF-Tu,SRP)and phosphatidylinositol signaling system(CALM,IMPA)in Chlorella-Arc were up-regulated under low temperature.Enhanced protein synthesis and its own complex signal transduction are conducive to alleviate cell damage under external stress.Under high temperature/low temperature stress,antioxidant enzymes(SOD,CAT)and key enzymes of GSH system(GSR,GPX,GST),were co-up-regulate,which can alleviate the oxidative damages induced by temperature stress and maintain the normal growth of cells.7.The Chlorella-Arc polysaccharide was extracted by conventional hot water extraction method.The optimal conditions(water ratio:48:1,extraction temperature 80?,extraction time 3 h)for extracting Chlorella-Arc polysaccharides were determined by response surface methodology(RSM),and the highest extraction rate of the polysaccharide was 9.61±0.11%.Three fractions(P-? P-?,and P-?)were presented after purification by 2-diethylaminoethanol Sepharose Fast Flow(DEAE Fast Flow)and Sephadex G-100 chromatography.The P-?a fraction demonstrated significant antioxidant activities than others at 5 mg/mL,with the scavenging effects on DPPH radicals,hydroxyl radicals,superoxide radical being 60.20±1.20%,72.10±1.50%,42.20±1.60%,respectively.Purity detection indicated that P-?a contains little proteins or nucleic acids.The major monosaccharide of P-?a was galactose,arabinose,rhamnose,and glucose.The molar ratios of galactose:arabinose:rhamnose:glucose was 3.67:1.08:0.66:0.38.Sulfate-group content analysis showed the presence of sulfate in P-?a with the content of 11.58±0.17%.The FTIR and NMR spectra showed that P-?a contained both ?-and ?-shaped contiguous sugars on the structure. |
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