Characterization And Functional Analysis Of The Transcription Factor MADS-Box EgAGL9 From Oil Palm(Elaeis Guineensis Jacq.) Mesocarp

Oil palm(Elaeis guineensis Jacq.)is one of the most important oil crops in the world,and it is also the highest productive efficiency oil crops.It is known as the World Oil King.Oil palm is native to tropical regions of Africa and is currently distributed in tropical regions such as Taiwan and Hainan.As an ancient and huge gene family,MADS-box transcription factor plays an important role in every stage of higher plants growth and development.In this study,based on the previously available differentially expressed gene data for the three different developmental stages of oil palm fruits,an MADS-box transcription factor of the AGAMOUS class was selected and named EgAGL9,which belongs to the type II MADS-box gene family.EgAGL9 was used as the research object in this study.Through bioinformatics analysis,ChIP-seq,construction of plant expression vectors,genetic transformation of oil palm embryoid callus,lipid analysis and other methods,the molecular mechanism of EgAGL9 regulating downstream genes was clarified.The results were as follows:1.EgAGL9,which gene length is 729 bp,was successfully cloned from the mesocarp RNA in oil palm fruits.Bioinformatics analysis showed that the MADS-box domain of EgAGL9 is highly conserved and may perform similar functions to other AGAMOUS-like MADS-box family genes.Real-time quantitative PCR results showed that the expression of EgAGL9 continued to increase during the later stages of oil palm fruit development.p35MK1-EgAGL9,a plant expression vector containing a 35s-specific promoter was constructed and found to be localized in the nucleus.The p GBKT7-EgAGL9 yeast expression vector also had been constructed,and it was found that EgAGL9 has transcriptional activation activity and has the characteristics of transcription factors.2.Three downstream genes EgSAD,EgTSA,EgSDH regulated by EgAGL9 were successfully screened by ChIP-seq.The results of EMSA showed that EgAGL9 could bind to motif in the promoter regions of EgSAD,EgTSA,and EgSDH,respectively.3.The plant expression vector p CAMBIA1300s-35s-EgAGL9 was constructed,and it was transferred into oil palm embryoid callus by Agrobacterium tumefaciens.After screening and identification,the relative expression of EgAGL9 in positive transgenic embryoid callus was more than 30,000 strains when it was compared to the wild type embryoid callus.The real-time quantitative PCR results of stable overexpression in oil palm embryoid callus and transient transfection of oil palm protoplasts showed that when the expression level of EgAGL9 increased significantly,the expression level of EgSAD decreased significantly,the expression level of EgTSA decreased significantly,and the expression level of EgSDH was declining.4.The total lipids of transgenic EgAGL9 embryoid callus were extracted and analyzed by GC-MS after methylation.The results showed that the total lipid content in the wild type embryoid callus was about 2.53 times that of transgenic EgAGL9 positive embryoid callus.The relative content of different types of fatty acids also changed.Compared with the wild type,the relative content of stearic acid(C18: 0),C20: 1,C20: 2 in the transgenic embryoid callus lines increased significantly,while the contents of oleic acid(C18: 1),linoleic acid(C18: 2)and linolenic acid(C18: 3)are significantly reduced.In summary,EgAGL9 interacts with the motif of the promoter regions of EgSAD,EgTSA,and EgSDH in the oil palm mesocarp.EgAGL9 regulates the expression of EgSAD,EgTSA and EgSDH through experiments such as stable overexpression of EgAGL9 embryoid callus,protoplast transformation,and real-time quantitative PCR.The results of this experiment preliminarily clarified that EgAGL9 regulates the relative content of oleic acid(C18: 1)by inhibiting the expression of EgSAD and thus affects the metabolism of unsaturated fatty acids in oil palm,affects the content of indole by inhibiting the expression of EgTSA and then regulates the metabolism of hormones,by inhibiting the expression of EgSDH regulates the TCA cycle to cut down the total oil content,which provides a certain basis for the subsequent elucidation of metabolic pathways and regulatory mechanisms research foundation.