Cloning And Functional Analysis Of The MxCYP82D47 Gene And Its Upstream Promoter In Malus Xiaojinensis

Iron(Fe)is an essential micronutrient for plant growth and development,yet very little Fe can be absorbed and used during plant growth and development.Therefore,elucidating the molecular physiological mechanisms of Fe deficiency tolerance in plants is of great relevance for improving Fe nutrition in crops through genetic means and genetic breeding.We screened the c DNA library of iron-deficient roots of the iron-efficient genotype Malus xiaojinensis for the MxCYP82D47 gene,which belongs to the CYP450(Cytochrome P450,CYP450)gene family of plant cytochromes.Cytochrome P450 is widely found in nature and is a heme monooxygenase encoded by a superfamily of genes.Cytochrome P450 enzymes are widely involved in plant secondary metabolism and plant resistance to stress,but the regulatory function of this gene in plant iron nutrition is not known.In this paper,we used Malus xiaojinensis and the model plant Arabidopsis thaliana as research objects,and conducted preliminary studies on the MxCYP82D47 gene of Malus xiaojinensis using plant physiology and molecular biology,and the main results are as follows:1.Tissue-specific analysis of the MxCYP82D47 gene in Malus xiaojinensis using real-time fluorescence quantification showed that MxCYP82D47 was expressed in all tissues,with significantly higher expression in young leaf tissues than in other parts.The MxCYP82D47 gene also responded to iron deficiency stress to varying degrees,and compared with normal culture CK,the MxCYP82D47 gene in young leaves of Malus xiaojinensis after iron deficiency stress treatment The expression level of MxCYP82D47 gene in young leaves of Begonia rose rapidly after the treatment of Fe deficiency compared with normal culture CK,reaching a maximum at 3 d of Fe deficiency,while the expression level in mature leaves increased significantly at the second and ninth days of Fe deficiency.However,the gene did not respond significantly to Fe stress in the roots.Thus,we tentatively concluded that the MxCYP82D47 gene responded to Fe stress and was induced to be expressed.2.Bioinformatics analysis of the CDS and promoter sequence of the successfully cloned MxCYP82D47 gene in Malus xiaojinensis showed that the full length of the gene was 1581 bp,encoding 394 amino acids.A phylogenetic tree was constructed to show that the gene with the highest homology to Malus xiaojinensis MxCYP82D47 is the Pyrus x bretschneideri P450 gene Pb CYP82D47.The MxCYP82D47 protein has a molecular weight of 59.05 k Da and an isoelectricity(p I)of 8.65.The results of subcellular co-localization experiments with the P1300-MxCYP82D47-YFP plant binary expression vector showed that the gene was localized in the endoplasmic reticulum.3.The upstream promoter of the MxCYP82D47 gene of Malus xiaojinensis was cloned at a sequence length of 2000 bp,and the promoter cis-element was predicted using the online software Plant CARE,showing that in addition to a large number of TATA-box and CAAT-box core promoter regions in the promoter of the MxCYP82D47 gene,there were also phytohormone-related In addition to the TATA-box and CAAT-box core promoter regions,there are also phytohormone-related elements,stress-inducible regulatory elements and light-responsive elements in the promoter of MxCYP82D47.The promoter activity was investigated by truncating the full length of the promoter according to the distribution of the cis-elements of the promoter.and MxCYP82D47-5P(-323 bp)five deletion fragments and fused to the P1300-LUC vector.The results of the dual luciferase reporter gene activity assay showed that the promoter was the most active in driving LUC gene expression at its full length,and there was a core promoter functional region between-771 bp and-323 bp.4.P1300-35S-MxCYP82D47 plant binary expression recombinant vector was constructed and MxCYP82D47 overexpression transgenic strain was obtained by transgenic technology.Iron deficiency stress analysis showed that the MxCYP82D47 overexpression transgenic line was less susceptible to green loss and more tolerant to iron deficiency stress than the wild type,and that iron deficiency stress significantly induced inter-root acidification,accumulation of trivalent iron reductase activity and more iron enrichment in the overexpression line.Furthermore,the expression of iron uptake related genes was induced at the transcriptional level to improve plant tolerance to iron deficiency stress.It is suggested that the MxCYP82D47 gene is regulating the response of Arabidopsis to iron deficiency stress.The results of this study provide preliminary evidence that the MxCYP82D47 gene of Malus xiaojinensis is a gene that positively regulates iron deficiency stress in Arabidopsis thaliana,which provides basic information for further analysis of the function of the MxCYP82D47 gene of Malus xiaojinensis and further exploitation of MxCYP82D47,and provides new genetic resources for genetic improvement of iron nutrition in plants.However,the mechanism of the MxCYP82D47 gene under Fe stress needs to be further investigated.