| Carbon allocation in microalgae can be changed under certain environment,which would result in the accumulation of some kind of carbon-based compounds in the cells.However,the exact mechanisms are unclear.Our previous studies have demonstrated that in the process of accumulation in chlorella under nitrogen-deficiency,the up/down multiple of the gene expression of one copper amine oxidase isoform 2(ChlCAO 2)is the highest.Copper amine oxidases(CuAOs)are a group of amine oxidases with various physiological functions,which participate in plant cell differentiation and response to abiotic stress,as well as the formation and differentiation of mammalian adipocytes.However,the physiological effects of CuAOs in Chlorella have not been reported.In order to further elucidate the role of ChlCAO2 in lipid accumulation induced by nitrogen deficiency,the correlation between copper amine oxidase and lipid accumulation was investigated.CuAOs activity and the relative expression of CHlCAO2 gene at 0,24,48,72,and 96h under three nitrogen level conditions(sufficient nitrogen,limited nitrogen and nitrogen recovery after nitrogen deficiency)in Chlorella sp.HN11 were studied.Furthermore,the transformed Chlorella sp.HN11 was obtained by genetic transformation of ChlCAO2 overexpression vector and RNAi vector,the phenotypes of lipid accumulation and ChlCAOs activity of transformed algae were determined in order to explore the role of CuAO in lipid accumulation induced by nitrogen deficiency in Chlorella.In addition,in order to find out whether the role of CuAO in lipid accumulation is common,the correlation between lipid accumulation and CuAOs was further examined in the other two green microalgae.Activity of CAOs in C.vulgaris C3 and Chlamydomonas reinhardiii CC425,and the relative expression of ChlCAO2 gene in C.reinhardtii CC425 under three nitrogen treatments were determined.The results showed that there was no lipid accumulation in Chlorella sp.HNll under sufficient nitrogen condition for 96 hours.The relative lipid fluorescence intensity was 3.03,4.48,4.17 and 4.63 times as high as that in control at 24h.48h,72h and 96h.The relative gene expression was 105,397,808,and 333 times as high as that in control,and ChlCAOs activity was 3.73,7.15,6.37,and 13 times as high as that in control.The fluorescence intensity of algae cell lipids decreased and cell density increased significantly after 48 hours of nitrogen limitation and then recovered from nitrogen supplementation(adding 0.1 g/L sodium nitrate)(P<0.05),the relative expression of ChlCAO2 decreased to 6.86%and 0.79%at 72 and 96 hours,and ChlCAOs activity decreased to 70.6%and 51.3%respectively.Copper amine oxidase activity and gene expression in Chlorella HN11 were positively correlated with lipid accumulation.No lipid accumulation was observed in C.reinhardtii cultured under sufficient nitrogen condition for 96 hours.The relative lipid fluorescence intensity was 3.17,4.49.7.58 and 5.15 times that in control,and CuAOs activity was 3.17,4.49,7.58 and 5.15 times that in control at 24,48,72,and 96 h respectively.The relative expression of CuAO1 was 236,721,348 and 1817 times that in control,the relative expression of CuAO02 was 11,4.7,13 and 12 times that in control,and the relative expression of CuAO3 was 1.64,2.11,5,and 11.5 times that in control at 24,48,72,and 96 h,respectively.The fluorescence intensity of algal cell lipids decreased and cell density increased significantly after 48 hours of nitrogen limitation and then recovered from nitrogen supplementation(adding 0.4g/L NH4CI)(P<0.05).the expression of CuAO decreased rapidly at 24 h and 48 h after nitrogen recovery.CuAO1 expression decreased to 0.04%and 0.22%,CuAO2 expression decreased to 0.67%and 17.7%,CuAO3 expression decreased to 3.04%and 24.83%,and CuAOs activity decreased to 23.4%and 25.2%,respectively.Activity of copper amine oxidase and gene expression in C.reinhardtii was positively correlated with oil accumulation.C.vulgaris C3 did not accumulate lipid during 96 hours under sufficient nitrogen condition.The relative lipids fluorescence intensity was 3.4,10.33,9.54 and 12.04 times that in control,and CuAOs activity was 1.97,2.46,4.3 and 5.62 times that in control at 24h,48h,72h,and 96h,respectively.The fluorescence intensity of algae cell lipids decreased and cell density increased significantly after 48 hours of nitrogen limitation and then recovered from nitrogen supplementation(adding 0.1 g/L sodium nitrate)(P<0.05),and CuAOs activity decreased to 95.8%and 35.7%at 24 h and 48 h after nitrogen recovery.CuAOs activity in C.vulgaris C3 was positively correlated with lipid accumulation.The full-length sequence of ChlCAO2 was obtained by RACE,which matched the sequence of transcriptome.The protein contains two domains,a Cu_amine_oxid N2 domain and a Cu_aminc_oxid domain,which exist in many other common species.Phylogenetic tree analysis showed that the amino acid sequence of ChlCAO2 had 56%homology with the copper amine oxidase in Chlorella sorokiniana and 54%homology with the copper amine oxidase in Micractinium conductrix.The overexpression and RNAi vector of ChlCAO2 were successfully constructed,and the plasmid vector was transformed into Chlorella sp.HN11 by Agrobacterium·mediated method.The growth of algae cells of C15,C21 strain with ChlCAO2 overexpressed and R13 strain with ChlCAO2 knockdown was not significantly different from that of wild type algae,the algae strains by transformed copper amine oxidase gene grew normally.The content of neutral lipids in C21 and C15 strain with ChlCAO2 overexpressed were significantly higher than that of wild type Chlorella(P<0.05),which were 1.6 and 1.93 times higher than that in control,respectively.ChlCAOs activity of C15 and C21 was significantly higher than that of wild Chlorella(P<0.05),which was 2.91 and 3.07 times higher than that in control respectively.The relative lipid fluorescence value in strain R13 was not significantly different from that in control.However,ChlCAOs activity of transformed algae strain R13 was significantly lower than that in control,which was 24%of that of normal cultured wild algae strain.These results indicate that ChlCAO2 may play a key role in lipid accumulation in Chlorella under nitrogen limitation conditions. |
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