| Chlamydomonas reinhardtii is a kind of single–celled eukaryotic green algae with a central chloroplast that occupies about 2/3 of the internal volume of the cell and carry out photosynthesis.Its huge chloroplast is unmatched by other higher plants.C.reinhardtii,as one of the dominant algae species for biological hydrogen production,has great potential for development.The main reason for its hydrogen production is the induced synthesis of hydrogenase,which is extremely sensitive to oxygen.At present,the method of hydrogen production is to reduce the photosynthetic oxygen evolution activity of C.reinhardtii through S-deficient culture,accelerate the establishment of anoxic environment in the culture system,induce hydrogenase expression,and increase hydrogen production.Existing studies have shown that hydrogenase is expressed under copper-deficient or hypoxic conditions and is activated by CRR1(Copper response regulater1).The CTRs copper transporter family of C.reinhardtii has 4 members,CrCTR1,CrCTR2,CrCTR3 and CrCOPT1,which have not been thoroughly studied at present.The expression level of copper deficiency responsive genes are increased under copper deficiency conditions.The promoters of these genes all have CuREs motif.The transcription factor CRR1 interacts with its core sequence GTAC to initiate expression,and studies have analyzed hydrogenase promoter sequences that also have CuREs motif.Genome analysis shows that the metal ion balance mechanism of C.reinhardtii may be a synthesis of the mechanisms in animals,plants,and microorganisms.Copper ion is an essential for the growth of C.reinhardtii.In order to ensure the normal physiological functions of copper ion and avoid the toxicity caused by excessive amounts,C.reinhardtii develops a series of regulatory mechanism.Using C.reinhardtii as a model to carry out research can clarify the cellular biological functions of copper ion in depth.In this study,through the study of the C.reinhardtii CrCTR2 copper transporter,we understand the physiological function of the copper absorption system.And to study the effect of C.reinhardtii copper transporter on photosynthetic hydrogen production under copper-deficient and hypoxic conditions.The main results are as follows:1.Bioinformatics analysis shows that CrCTR2 has 3 transmembrane regions and the N-terminal sequences has special metal binding Cxx Mxx Mxx C motif.Yeast functional complementation experiments confirmed the function of CrCTR2 in transporting copper in yeast.Constructing GFP fusion expression vector,under confocal laser observation,CrCTR2 is localized on the cytoplasmic membrane in rice protoplasts.2.The growth curve,chlorophyll content and SOD enzyme activity of the CrCTR2 gene mutants and overexpression algae strains were measured under the copper treatment strip.It was found that under the copper deficiency treatment conditions,the growth of algae were inhibited.But compared to the wild-type and overexpression,the mutant Δctr2has better growth and lower SOD enzyme activity,but the overexpression is not significantly different from the wild type.3.Under the condition of hydrogen production in the absence of S,the gas chromatograph was used to draw 500 μL of gas above the culture flask with a micro gas sampler,and the hydrogen production in 11 days were detected.The results showed that the mutant strain rapidly produced hydrogen from the 4th to the 7th day,and the hydrogen production was higher than that of the wild type at the 7th day.Moreover,under the condition of further copper deficiency,the hydrogen production further increase on the 7th day.4.Analysis of the photosynthetic system Ⅱ(PSⅡ)activity of C.reinhardtii cells under sulfur deficient hydrogen production condition,it was found that the Fv/Fm and ETR of the mutant strain is lower than those of wild type,indicating that the mutant strain can further reduce the activity of PSⅡ.The PSⅡ of C.reinhardtii cells is further reduced under both sulfur and copper deficiency treatment,indicating that copper deficiency can also reduce PSⅡ activity.5.Further analysis of the expression level of HyDA1 hydrogen under hydrogen production conditions showed that its expression level show a trend of increasing first and then decreasing.However,under copper-deficient conditions,the level of hydrogenase expression maintained at a relatively stable level and the expression level is higher.At the same time,by analyzing the expression of the copper deficiency response coproporphyrinogen oxidase1(CPX1),the activity of CRR1 is analyzed,and it is found that the activity of CRR1 of the mutant stains was signification higher than that of control group on the 4th day.And under the condition of copper deficiency,the CRR1 activity of the mutant strain is higher than that of the control group from the first day.It shows that copper deficiency increase the activity of CRR1.6.The hydrogenase activity of the mutant strain of C.reinhardtii at different time points under hydrogen production are analyzed,and it is found that the hydrogenase activity of the mutant strain under S-deficiency is not significantly from that of the wild type in the first 4 days,but on the 7th day,the hydrogenase activity of the mutant strain is higher than that of the wild type.Further lack of copper,the activity of hydrogenase is further improved.In summary,the CrCTR2 copper transporter of C.reinhardtii is located on the cytoplasmic membrane.The heterologous expression of yeast proves that CrCTR2 has the function of transporting copper.In the S-deficient hydrogen production environment,the mutants Δctr2 has a certain promotion effect on the hydrogen production of C.reinhardtii,and the hydrogen production of mutant increased more significantly after further copper deficiency treatment.Under the condition of S and copper deficiency,the PSⅡ activity of C.reinhardtii is further reduced compared with the condition of single deficiency of S.The hydrogen production of C.reinhardtii from 7th day has been improved overall compared with that under the condition of single deficiency of S.The expression of hydrogenase and the activity of CRR1 proves that the lake of copper affects the activity of CRR1,activates the expression of hydrogenase,and increase hydrogen production.The hydrogen production of the mutant is higher than that of wild type,which may be because the copper transporter mutant Δctr2 of C.reinhardtii inhibits in the copper transport function,which reduces the copper content in the algae cells and stimulates CRR1 to play a role.The decrease of copper content in algae cells further induces hydrogenase under hypoxic conditions,which increase the content of hydrogenase in algae cells,thereby increase hydrogen production. |
