Cloning And Function Characterization Of TFIIE?ACBP4 And MAP70-1 Genes In Apple(Malus Domestica Borkh.)

In this experiment,the Apple Rootstock M26 was used as the material.On the basis of the sequencing of the early phosphorylation protein group,TFIIE?General transcription factor IIE subunit 1-like?,ACBP4?Acyl-CoA-binding domain-containing protein 4?and the differential protein were screened.TFIIE,ACBP4 and MAP70-1 genes were cloned by RT-PCR method,and the plant dual expression vector was constructed.Then the above three genes were successfully introduced into the WS Columbia Arabidopsis?Arabidopsis thaliana?using the Agrobacterium mediated method.Functional identification of TFIIE,ACBP4 and MAP70-1 genes was carried out under PEG treatment.The detail results are as follow:1.TFIIE,ACBP4 and MAP70-1 genes were cloned,and the accession numbers of genes were MG878870,MG878871 and MG878872 respectively.The size of TFIIE and ACBP4genes were 1413 BP and 2016 BP respectively.The homology of the amino acid sequence of the pear was the highest,which was 97.87%and 94.82%,respectively.The fragment size of MAP70-1 was 1860 bp,and the homology of MAP70-1 amino acid sequence from strawberry was 91.20%.2.The results of fluorescence quantitative PCR showed that the expression level of TFIIE in M26 pot seedlings was up-regulated while ACBP4 was down-regulated under continuous water control for 20 d,40 d and 60 d.There was no significant difference in the expression level of MAP70-1.3.The plant dual expression vector driven by the component promoter 35S was constructed,and Agrobacterium tumefaciens EHA105 was successfully transformed,named pBI121/TFIIE/EHA105,pBI121/ACBP4/EHA105 and pBI121/MAP70-1/EHA105.4.Transgenic T₂ was obtained from above three transgenic plants.After 15%PEG stress,TFIIE of transgenic Arabidopsis was up-regulated by 2.35-fold,and ACBP4 was down-regulated by 1/3.There was no significant difference between MAP70-1 and the control.The germination rates of T2 transgenic seeds of these three genes under PEG treatment were 90.3%,76.23%and 88.66%,respectively.In addition,the survival rates were 79.68%,28.30%and 54.35%,respectively.