Cloning And Preliminary Functional Analysis Of Several Ethylene Response Factors In Maize

Ethylene is a regulator of plant growth and development and plays an important role in different aspects of plant growth and development and stress response.In this study,ethylene treatment was performed on the self-pollinated 5 DAP ears for popcorn maize inbred line N04 and 20DAP kernels were selected for differential protein identification.The inbred lines N04 and Dan232 were used as materials to carry out the homologous cloning and sequence analysis of the ethylene receptors ZmETR2,ZmERS 14 and ZmERS25 and the ethylene response factor ZmMPK6.The homologous cloning of the promoter sequence of ZmETR2 and the prediction of the functional elements were further performed.A subcellular localization vector was constructed and subcellular localization prediction analysis was performed to construct an overexpression vector for genetic analysis of Arabidopsis thaliana.An expression vector for ZmMPK6 was constructed and subjected to genetic transformation analysis.The main results were as follows:1.From the maize inbred lines N04 and Dan232,a band of about 2500 bp for ZmETR2 was amplified and the open reading frame(ORF)was 2301 bp.The ORF sequences of inbred lines Dan232 and B73 were the same.There was one base difference between the ORF sequences of N04 and B73,but there was no difference in their encoded amino acid.It encoded 767 amino acids.Sequence analysis revealed that ZmETR2 had a typical ethylene receptor family structure and properties,which was a member of the ethylene receptor family.Evolutionary analysis showed that the conservation of ZmETR2 protein in wheat,rice,sorghum,soybean,millet,Arabidopsis and other species was low.2.The promoter region of ZmETR2 was cloned from maize inbred lines N04 and Dan232,respectively.There was no difference between the two inbred line.The predicted elements of the cloned promoter region revealed that the promoter region of the ZmETR2 contains a variety of light-responsive elements as well as response elements,such as stress and phytohormones and plant development.The subcellular localization vector of ZmETR2 was successful constructed and subcellular localization prediction showed that it was located on the endoplasmic reticulum.3.The sequences of ZmERS14,ZmERS25 and ZmMPK6 were obtained from the maize inbred lines N04 and Dan232 by homologous cloning.After analysis of the sequence obtained,it was found that the amino acid of ZmERS14 from N04 changed at position 590,resulting in a change in its domain,but no change was found in inbred Dan232.The sequence of ZmERS25 cloned from the inbred lines N04 and Dan232 showed no difference in their protein domains,although there were individual differences in the encoded amino acids.The sequence of ZmMPK6 was obtained from inbred lines N04 and Dan232.There was no difference between Dan232 and B73,but the cDNA sequence obtained in N04 was quite different.The insertion of the first 10 bp base intron of the second exon of the DNA sequence caused the ORF sequence shorter.Protein domain predictions found that its domain changed from the complete serine/threonine protein kinase domain(S_TKc)to the STYKc domain currently found and less annotated.4.Plant expression vectors 3301-ZmETR2,3301-ZmMPK6d and 3301-ZmMPK6c were constructed and successfully transformed into Arabidopsis thaliana.Plants transfected with the 3301-ZmETR2 have been grown to the T2 generation.Transgenic plants of 3301-ZmMPK6d and 3301-ZmMPK6c have obtained T0 generations.Further study should be conducted to reveal their functions.5.Ethephon spray treatment was conducted on the maize inbred line N04 on pollinated ears at 5 DAP.The 100-grain dry weight of the treatment group was significantly higher than that of the control group at 20 DAP.Accordingly,differential protein identification analysis was performed on the seeds of 20DAP.Totally,18 differential proteins were identified from 36 differential protein spots.These proteins were mainly involved in stress response,energy metabolism,protein post-translational modification,signal Transduction and protein synthesis and unknown function proteins.Among them,the identified differential proteins PPDK1 and PPDK2 were the rate-limiting enzymes of the C4 photosynthetic pathway,and they were key enzymes of the glycolytic pathway.It is speculated that the C4 photosynthetic pathway,glycolysis pathway and ethylene signal transduction pathway might link through PPDK and together regulate the grain filling.