Study On Apple Ethylene Response Factor ERF1B Regulating Aroma Biosynthesis In LOX Pathway

The effect of fruit aroma on apple quality is decisive.A good scent of the fruit can increase its commercial value and thus obtain greater commercial value.The synthesis of fruit aroma is relatively complicated.Among them,the lipoxygenase pathway is widely studied.As the key gene of this pathway,the lipoxygenase gene e has attracted scholars’ attention,but the specific mechanism has yet to be fully elucidated.Ethylene promotes the maturation of fruits and is called a fruit maturation hormone.In most plants,the ethylene response factor ERFs are located downstream of the ethylene response pathway,transmitting ethylene signals to downstream genes,completing the transduction of ethylene in fruit maturation.What’s more,ethylene participates in the aroma synthesis of fruits by regulating the expression of LOX genes such as PpERF1,PpERF2,etc.However,the mechanism of ERF family specific involvement in aroma synthesis in apples is unclear.Research on MADS protein has never stopped,especially in regulating ethylene signal transduction,participating in fruit maturation,and aroma synthesis.Therefore,in order to further explore the functions of mentioned families,we use ‘Changfu 2’ as a test material,through quantitative gene screening,clones MdLOX1 a,MdMADS24 and MdERF1 B genes,and studies the gene function.The results are as follows:1.Sampling every 15 days at 105 days after flowering of apples to determine the relative content of aroma substances and ethylene release rate;real-time fluorescence quantitative screening of structural genes of fatty acid synthesis aroma pathway,MADS and ERF families was performed.The results showed that as the fruit matured,the relative content of aroma substances and the release of ethylene showed an upward trend,and MdERF1 B,MdMADS24 and MdLOX1 a were significantly up-regulated.Next,we will use these genes as target genes for further exploration.2.MdLOX1 a was over-expressed to ‘Orin’.Compared with the control group,the aroma components of the experimental group increased to varying degrees,indicating that MdLOX1 a promotes the synthesis of aroma substances.In addition,analysis of the promoter element of this gene revealed that there is a cis-acting element of CArG-box,a binding element of the MADS family on the promoter.3.MdMADS24 was over-expressed in Apple Wang Lin.After transgenic verification,it was found that the aroma substance doubled and MdLOX1 a up-regulated the expression.Through yeast one-hybrid experiments,ChIP and EMSA experiments,it was found that MdMADS24 specifically recognized and combined with the CArG-box element on the MdLOX1 a promoter.LUC experiments confirmed that the MdMADS24 protein promoted the activity of the MdLOX1 a promoter.4.Co-transform MdERF1 B to OEMdMADS24 callus.After verifying the success of the transgene,determine the relative content of aroma substances and gene expression.The results show that compared with the single conversion of OEMdMADS24,the relative content of aroma substances in the co-transformed materials increased,Expression was significantly upregulated.The yeast two-hybrid(Y2H)experiment and pull down were used to verify the interaction between MdMADS24 and MdERF1 B proteins.The BiFC experiment was performed again in vivo to verify that the two did indeed interact with each other to produce a protein complex.The results of the LUC fluorescence report showed that the protein of MdMADS24 promoted the transcriptional activity of the MdLOX1 a promoter,and the complex of MdBBX24 and MdERF1 B proteins promoted the MdLOX1 a promoter The effect of activity is more significant.The results of the study indicate that MdMADS24 promotes the transcription of the latter by binding to the MdLOX1 a promoter,and at the same time MdMADS24 interacts with the MdERF1 B protein-protein downstream of the ethylene delivery pathway to promote the transcriptional activity of the MdLOX1 a promoter and thereby participate in the synthesis of aroma components in fruits.The molecular mechanism of ethylene indirectly participating in fruit aroma synthesis was revealed for the first time,providing a theoretical basis for ethylene participating in fruit aroma quality formation.