Cloning And Functional Identification Of NF-YA/B/C From Chlorella Ellipsoidea And The Effects Of Fe²⁺ Onlipid Synthesis Of Chlorella Ellipsoidea

Algae is considered to be one of the most promising source for producing biofuel.With the growing demand for edible oil and biodiesel,it is extremely urgent to search new organisms with potential high yield and environmental sustain ability for producing lipid.It is a feasible method to increase the oil content in oil crops by overexpressing transcription factor associated with lipid synthesis.Chlorella ellipsoidea grows rapid and can accumulate high content of lipid under low nitrogen conditions.NF-Y is a transcription factor with significant effect on seed development,lipid synthesis and accumulation.We tried to clone NF-Y genes from Chlorella ellipsoidea and characterize their functions in Arabidopsis thaliana to provide technical and theoretical basis for increasing oil content of oil crop such as oil seed rape.Using Chlorella ellipsoidea as experimental material,we cloned three transcription factor genes,NF-YA?CeNF-YA?,NF-YB?CeNF-YB?,NF-YC?CeNF-YC?.The amino acid sequences of CeNF-YA,CeNF-YB and CeNF-YC were compared with those from human,yeast,mouse,Arabidopsis thaliana,rice and oil seed rape.The similarities of the conserved domains were 56.6%-71.7%,62.9%-91.4%,35.1%-93.5%,respectively.We constructed the yeast expression vector pYDEST02 of these three genes and then they were expressed in yeast INVSc1.These three genes could be expressed in yeast,but the total fatty acid contents of transgenic yeast were not significantly changed.CeNF-YA,CeNF-YB and CeNF-YC were expressed in Arabidopsis thaliana by transferring their plant expression constructs,respectively,and the T1 transgenic plants were obtained.The 1000-seed weight of T1 lines of CeNF-YA,Ce NF-YB and CeNF-YC transgenic lines was significantly increased than that of wild type Col-0 plants by 26.5%-44.9%,24.5%-51% and 18.4%-44.9%,respectively.The content of fatty acid in T1 transgenic lines was increased by 22.4%-18.4%,7.1%-11.2 % and 9.8%-15.4%,respectively.Furthermore,the fatty acid composition of T1 lines was not changed much.However,the content of C18:0,C18: 1,C18: 3 and C20: 1,the main content of fatty acids composition,was significantly increased.The content of C18:0 was increased by 14.5%-24.4%,12.2%-24.5% and 1.9%-28% in Ce NF-YA,CeNF-YB and CeNF-YC transgenic lines,respectively;C18:1increased by 10.5%-31.0%,10.8%-29.9% and 1.1%-7.9%,respectively;C18:3 increased by 27.2%-35.2%,3.5%-6.4% and 22.6%-28.5%,respectively;C20:1 increased by 27.4%-34.8%,6.5%-20.6% and 7.6%-16.0%,respectively.In order to further study the molecular mechanism of CeNF-YA,CeNF-YB and CeNF-YC function,the subcellular localization of CeNF-YA,CeNF-YB and CeNF-YC were carried out and they were localized in the nucleus.The interaction experiment of CeNF-YA,CeNF-YB and CeNF-YC showed that CeNF-YA interacts with Ce NF-YB and CeNF-YB interacts with CeNF-YC.In addition,we also studied the effects of different concentrations of Fe²⁺ on Chlorella ellipsoidea growth,lipid accumulation,and the expression of genes involved in lipid synthesis.The results showed that the condition of high concentration of Fe²⁺ promotes the growth of Chlorella ellipsoidea.Fe²⁺ deficiency is beneficial to the accumulation of fatty acids,and up-regulates the expression of CeNF-YA,CeNF-YB and CeNF-YC.The expression of CeNF-YA,CeNF-YB and CeNF-YC could be helpful to the improvement of fatty acid content.Our study provides new clues to elucidate the functions of CeNF-YA,CeNF-YB and CeNF-YC,and provides potentially valuable gene resources to improve the oil content of oilseed crops by transcription factor.