Cloning,functional Analysis And Regulation Mechanism Of CoFBA From Coconut(Cocos Nucifera L.)Endosperm

Coconut(Cocos nucifera L.),an important crop in the tropics,is the only species of the Cocos Palmae.Fructose-1,6-bisphosphate aldolase(FBA)is a key enzyme in glycolysis of higher plants.In order to study the function of CoFBA gene in coconut during endosperm oil accumulation and analyze their regulatory mechanisms in the oil metabolism pathway,CoFBA were first cloned and overexpressed in Arabidopsis thaliana,and transcription factor GATA2 interacting with its promoter is screened.The function of CoFBA and the regulation of CoFBA by GATA2 were elucidated via vector construction,transgenic,fatty acid extraction and EMSA.The achieved results are as follows:1.The gene CoFBA was cloned from coconut and constructed into p CAMBIA1300 s,a vector driven by seed-specific promoter napin,to investigate the effect of overexpression of CoFBA in Arabidopsis thaliana.The contents of total oil and fatty acid were analyzed in three transgenic lines with different CoFBA transcript levels.Compared with the wild type,the oil content of the seeds of three transgenic lines increased significantly(3.5%-18%),but there was no significant difference among the lines.Moreover,the contents of linolenic acid(C18:3)and eicosanoic acid(C20:1)increased significantly in transgenic plant seeds,which indicated that CoFBA overexpression not only increased the total lipid content but also altered the fatty acid composition in transgenic lines.2.The promoter of CoFBA was cloned from coconut by using the DNA of leaves as template.Its sequence analysis showed that the promoter contained a large number of cis-acting elements.The yeast expression vector p HIS-pro FBA was transferred into coconut c DNA library by yeast one-hybrid experiment and screened a transcription factor GATA2.The GATA2 was ligated into plant expression vector p35MK1-35S-GFP for subcellular localization experiment and found localized in the nucleus,thus verifying GATA2 is likely to have transcription factor activity that can regulate the expression of CoFBA.3.Prepared the protein expression vector p COLD-GATA2 and purified the protein.The result of EMSA verified the transcription factor GATA2 was able to bind to pro FBA,and the interaction between them was affirmed by the one-to-one plate experiment.Recombinant vector p GreenⅡ62-SK-GATA2 was transferred into coconut protoplasts,then the expression of CoFBA decreased.It suggested that transcription factor GATA2 inhibit the expression of CoFBA.In summary,gene CoFBA in coconut can increase oil content and change its fatty acid composition in transgenic Arabidopsis thaliana seeds;the transcription factor GATA2 screened by yeast single hybrid can interact with CoFBA promoter and inhibit its expression;these results provide a feasible way to study the metabolic pathway of oil in coconut and the molecular regulation mechanism of genes related to the metabolism of liquid.