Screening And Regulatory Mechanism Analysis Of Transcription Factor Interacting With Fatty Acid Desaturase(FAD2) Promoter In African Oil Palm(Elaeis Guineensis Jacq.)

Oil palm(Elaeis guineensis Jacq.),named as oil coconut,is a perennial monocotyledonous evergreen tree.The oil palm is the world's most valuable oil crop,the oil content in per hectare is 6-11 times in comparison with rapeseed and soybeans.Oil palm is wide-spread occurrence in Africa,Southeast Asia,South America,and Hainan Province in China.The mesocarp is rich of oil,which accounts for the total weight about80%.The fleshy fruit mesocarp contains approximately equal proportions of saturated fatty acids and unsaturated fatty acids,and in which the ratio of oleic acid and linoleic acid is about 4:1.Linoleic acid is an essential fatty acid,used as a substrate to synthesize biological active substances to maintain the normal physiological metabolism of the organism.The oleic acid desaturase FAD2(?12 Fatty acid desaturase),can dehydrogenate oleic acid to form linoleic acid,which plays an important role in regulating the ratio and content of oleic acid and linoleic acid.In this study,in order to analyze the regulatory mechanism on Elaeis guineensis FAD2(Eg FAD2)in fatty acid metabolism,the promoter Eg FAD2 has been cloned and its interacting transcription factors has been screened and their regulatory relationship has been clarified.The research results obtained are as follows:1.Based on the previous results,sequence of Egpro FAD2 promoter has been analyzed and found that Egpro FAD2 contains various cis-acting elements,such as hormone response elements,light response elements and others by bioinformatics analysis.2.The plant expression vector Egpro FAD2: GUS has been constructed,and then infecting into Arabidopsis thaliana.Positive transgenic plants have been obtained and staining the GUS protein.Results showed that GUS protein can be expressed in all tissues of Arabidopsis thaliana,without obvious specificity among tissues.3.Yeast one-hybrid was employed and a total of 94 proteins were screened out.Among them,Eg ARF18 was selected to further study since ARF family genes has been reported involved in the growth of flower organs and seeds in plant.Result of quantitative real-time PCR(q RT-PCR)analysis showed that the expression of Eg ARF18 was highest in the fourth stage(120-140 days after flowering)during the oil palm fruit development,and this result was in consistence with the expression tend of Eg FAD2.4.Results of bioinformatics analysis show that Eg ARF18 protein,which containing the B3 domain and the Auxin?resp conserved domain,was predicted to be located on the nucleus.Localization experiments were performed and further confirmed that Eg ARF18 localized in the nucleus.5.Eg ARF18 protein has been heterologously expressed and purifie.Electrophoretic mobility shift assay(EMSA)was employed to demonstrate the interaction-ship between Eg ARF18 protein and the sequence of promoter Eg FAD2.As results shown,Eg ARF18 can bind to the auxin response element,named Aux REs,the sequence of TGTCXX on the promoter Eg FAD2.6.Eg ARF18 gene has been transformed transiently into oil palm protoplasts and transgenic Arabidopsis thaliana.q RT-PCR analysis was carried out and results reveal that the expression level of Eg FAD2 was significantly up-regulated with the overexpression of Eg ARF18 in transgenic oil palm protoplasts,when compared with the wild type(WT).Similarly,it has been found that the content of GUS protein was significantly increased in the overexpressed-Eg ARF18 transgenic Arabidopsis leaves compared to not overexpression.7.Construct of the stably transgenic oil palm embryogenic callus with overexpressed-Eg ARF18 and q RT-PCR analysis was employed to determined the expression level of Eg FAD2 and Eg ARF18 in the transgenic oil palm embryogenic callus.Significantly,results showed that the expression level of Eg FAD2 and Eg ARF18 were upregulated in transgenic oil palm embryogenic callus and both of them exhibited positive correlation.It has been deduced that Eg ARF18 has some regulatory functions on the expression level of Eg FAD2.Above all,Eg ARF18,a transcription factor,was screened and found interacting with Eg FAD2 in oil palm.Eg ARF18 can directly bound to the cis-acting element TGTCXX on the promoter of Egpro FAD2 and promoted Eg FAD2 expression.It has been suggested that Eg ARF18 was an vital transcription factor,which play a regulatory role on linoleic acid biosynthesis in oil palm.These results would provides a powerful molecular basis for the creation of new genetically improved varieties with great content of linoleic acid.