Expression And Function Analysis Of An Upstream Regulation Sequence Of Citrus Junos Ferric Reductase Oxidase Gene (CjFRO1) In Tobacco

Plant chlrosis was a world wide problem of plant malnutrition,iron-deficiency could caused leaf chlrosis and harmed the yield of plant.The FRO2 gene,first found in Arabidopsis and detected in root,could be induced to express under the environment of iron deficiency.Luo et al.（2007） cloned the CjFRO1 gene from Citrus junos,which conferred the tolerance to the iron-deficient environment when introduced into Poncirus trifoliate.The result revealed that CjFRO1 gene has the function of tolerating iron deficient environment.To determine the CjFRO1 gene’s expression characters in plant, cloning and further research its 1824bp upstream regulation sequence of CjFRO1,five different length promoters fused to aβ-glucuronidase（GUS） reporter gene expression-vectors were constructed by 5’ terminal deletion.Based on the data of comparison of cis-acting elements with database（Plantcare）,genetic transformation of tobacco（Nicotiana cv Xanthi）,different tissues histochemical staining analysis and expression profiles analysis,the main results as below:1.Five different length promoters fused to a GUS reporter gene were introduced into tobacco plants.The results of PCR amplification validated and histochemical staining analysis showed that GUS reporter gene can be detected when the promoter sequence was no shorter than 1342bp. 2.Expression profiles analysis of 1342bp,1515bp and 1824bp promoter transgenic tobacco revealed that GUS gene can be detected in leaf,stem and root,and most remarkable in leaf.No marked difference GUS gene expression between 8mmol/L Fe³⁺ and Fe²⁺ treatment.3.Analyzed the GUS activity of different growth stages leaves from 1824bp promoter transgenic tobacco,the results showed that the highest GUS activity in mature leaves,the GUS activity in senescence leaves and shoot just reached about half of mature leaves.4.Histochemical staining of transgenic tobacco leaves with dark treatment showed negative,but positive from leaf with normal light environment.It suggested that the light-regulatory elements,such as G-Box,TCT-motif,had function on expression regulation and CjFRO1 gene expression may be light-depended.Experiment data showed that CjFRO1 gene promoter expresses mainly exist green tissue,otherwise express in root slightly,be barely induced by iron deficiency,suggests that light-regulatory elements may have crucial influence on efficiency of promoter.