| Oil palm(Elaeis guineensis Jacq.)is the highest productive efficiency of oil crops in the world.It is known as the "world oil king" and is mainly distributed in Taiwan,Hainan and Yunnan area in China.Diacylglycerol acyltransferase(DGAT)is the last step in the synthesis of Triacylglycerol(TAG)and the only rate-limiting enzyme in this pathway.In order to analyze the regulatory mechanism of DGAT2 promoter in the oil palm lipid metabolic pathway,the transcription factors interacting with DGAT2 promoter were screened and identified.DGAT2 promoter and MADS21 were used as the research objects,and DGAT2 promoter was clarified by transgenic,vector cloning,bioinformatics analysis and fluorescence quantification.The tissue expression of the promoter and the effect of MADS21 on DGAT2 expression were as follows:1.promoter region of DGAT2 gene was isolated and characterized from oil palm.Based on genomic DNA sequence analysis,1035 bp promoter region was amplified by PCR.Several kinds of functional elements,such as TATA-box and CAAT-box were identified from the obtained sequence by alignment in database.In order to verify the GUS gene’s tissue specificity in different tissues,constructed plant expression vector promoter-DGAT2-pCAMBIA1304 and transformed the vector into Arabidopsis thaliana via the floral dip method.GUS staining of the transgenic Arabidopsis indicated that DGAT2 promoter can normally drive report gene expression without significant tissue specificity.2.Preparated yeast expression vector promoter-DGAT2-pHIS2.1 and transferred into Y187 yeast containing the oil palm library.54 proteins interacting with the DGA T2 promoter were screened,based on previous research,MADS-box family play prominent role for plant growth and flower development.Therefore,MADS21 was selected as the main research target,the MADS21 gene was amplified by PCR,the full length was 781 bp,the results of bioinformatics analysis indicated that it belonged to the II-type MEF-like of MADS-box family.The results of RT-PCR showed that the expression level of MADS21 decreased in the last two periods of oil palm fruit development,which was basically opposite to the transcription level of DGAT2.3.Biotin-labeled the SRF binding site in the DGAT2 promoter sequence,while constructing a protein expression vector MADS21-pCOLD to obtain the purified protein.The interaction of MADS21 and DGAT2 promoter can be verified by Electrophoretic Mobility Shift Assay(EMSA).Constructed the plant expression vector MADS21-1300s and transferred into protoplast:after transfecting into MADS21 in transgenic Arabidopsis protoplasts,the activity of GUS protein was significantly decreased,that is to say MADS21 could bind to DGAT2 promoter and its efficiency was decreased;after transferring MADS21 in oil palm protoplasts,the quantitative expression of DGAT2 was significantly decreased by RT-PCR,indicating that MADS21 inhibited the expression of DGAT2.In addition to transient expression,RNA was extracted from oil palm callus material stably expressing MADS21.The fluorescence quantitative results showed that the expression of DGAT2 was significantly decreased after MADS21 overexpression,and the higher the expression of MADS21.DGAT2 expression was inhibited severely.In summary,DGAT2 promoter can normally drive report gene expression without significant tissue specificity,and there is a significant interaction between MADS21 and DGAT2 promoter by one-hybrid screening,through protoplast transformation,fluorescence quantification,etc.The experiment can prove that MADS21 inhibits the expression of DGAT2.After the stable expression of MADS21,the expression of DGAT2 is extremely significantly inhibited.The results of this experiment preliminarily clarified that MADS21 regulates the lipid metabolic pathway of oil palm by inhibiting the expression of DGAT2,which provides a basis for the subsequent clarification of metabolic pathways and regulatory mechanisms. |
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