Analyzed by qRT PCR Fig the variation of the
Analyzed by qRT-PCR (Fig. 8), the variation of the expressions of DtACLA and DtACLB was fairly consistent in response to nitrogen deficiency, suggesting that DtACLA and DtACLB may be coordinate to function in the catalysis. It was reported that in Arabidopsis, the coordinated ACLA and ACLB mRNA accumulation pattern is coincident with the accumulation pattern of acetyl-CoA carboxylase, an enzyme using cytosolic acetyl-CoA as a substrate . It was indicated that both subunits of ACL from C. tepidum contribute to the active site of ACL . It was reported that lipids accumulated in D. tertiolecta under nitrogen deficiency condition by days 3–7 . In this study, it was found that the expressions of DtACLA and DtACLB under nitrogen deficiency condition were increased and coincident with the moment of lipid accumulation mentioned by Chen et al. . The highest transcript levels of DtACLA and DtACLB happened at the nitrogen-deficient treatment by day 7 and day 5. It seemed that the expressions of DtACLA and DtACLB was related to the lipid accumulation in D. tertiolecta. It was reported that the expression levels of ACS from D. tertiolecta were increased after nitrogen starvation cultivation, indicating that ACS activity may be also linked to the lipid accumulation .
By using microwave assistance to extract lipids from D. tertiolecta FACHB-821, the lipid content reached to be 57.02%, indicating that D. tertiolecta is considered as a potential biodiesel feedstock . The biomass production and lipid accumulation in D. tertiolecta can be limited by several factors, such as nutrients. It was reported that lipids production from D. tertiolecta could be increased under nitrogen starvation or other nutrients such as iron and cobalt, while phosphate deprivation had little effect on lipid accumulation . Lee et al. reported that much more fatty acids production was observed by shifting the culture condition to nitrate deficiency and high light at exponential phase . It might be applicable to fatty CVT 10216 production by the optimization of cultivation conditions for the use of D. tertiolecta as a biofuel source. However, it was suggested by Mata et al. that residual wastewaters rich in nitrogen (N) and iron (Fe) could be used to cultivate D. tertiolecta for lipid production, because N and Fe addition to the culture medium resulted in a significant increase in lipid productivity . With the help of next-generation sequencing, the transcriptome sequencing and annotation of D. tertiolecta UTEX LB 999 were reported , which can provide a valuable resource for identification of genes involved in biosynthesis and catabolism of fatty acids, TAG, and starch in D. tertiolecta. By providing insight into the mechanisms of these metabolic processes, genetically manipulation of D. tertiolecta can be used to enhance the production of feedstock for commercial microalgae-biofuels.
In summary, genes encoding the two distinct subunits (ACLA and ACLB) from unicellular alga D. tertiolecta have been cloned and characterized. TFBSs predicted in the 5′-flanking regions of DtACLA and DtACLB should be identified by further experiments, so that the regulation mechanism of ACL can be elaborated. The expressions of DtACLA and DtACLB were fairly consistent with lipid accumulation in response to nitrogen deficiency, suggesting that both subunits of ACL from D. tertiolecta contribute to the active site of ACL and the ACL activity was related to lipid accumulation in D. tertiolecta. Further study on the relationship between nitrogen requirements and lipid productivity of D. tertiolecta is in progress.
Acknowledgements This project was supported by the National Natural Foundation of China (31171631 and 31571773), Guangdong Province Science and Technology Plan Project (2011B031200005), and Guangdong Provincial Bureau of Ocean and Fishery Science and Technology to promote a special (A201301C04).
Introduction Polyamines (i.e., putrescine, spermidine, and spermine) are essential for cell proliferation and regulation of various cellular processes . High concentrations of cellular polyamines have been reported in various types of cancer . Antizyme (AZ) is a negative regulator of cellular polyamines. AZ is induced by polyamines through a unique mechanism, translational frameshifting . AZ binds to ornithine decarboxylase (ODC), a key enzyme in polyamine synthesis, and accelerates the degradation of the enzyme protein by the 26S proteasome . AZ also inhibits the cellular uptake of polyamines . By blocking both the biosynthesis and uptake, AZ quickly and strongly suppresses the cellular polyamine supply. AZ has also been shown to promote polyamine excretion . The overexpression of AZ has been shown to lead to growth inhibition of cells and exerts an anti-tumor activity . Three types of AZ exist in mammals: AZ1 and AZ2 are expressed in most tissues, whereas AZ3 is observed only in the testis . The distribution of AZ1 and AZ2 overlaps, and the expression level of AZ1 is much higher than that of AZ2 . Recent studies have reported that AZ1 also binds to and accelerates the degradation of other functional proteins such as Smad1, cyclin D1 and Aurora-A , , . Another AZ-binding protein is antizyme inhibitor (AZI), an ODC-related protein, which binds to AZ in competition with ODC . The binding of AZ stabilizes AZI .