Cloning And Functional Analysis Of The Ferric Reduction Oxidase Gene In Maize(ZmFRO2)

Fe is at a low level in plants but is efficient for their normal growth and good development.In crop breeding,increasing Fe content in crops and grains is of great significance for improving crop quality and crop yield.Existing in Arabidop.sis thaliana,Lycopersicum esculentum,Glycine max,Oryza sativa,Arachis hypogaea and Tribulus terrestris and other higher plants,FRO(Ferric Reduction Oxidase)family genes were studied.This study used AtFRO2 amino acid sequence as probe to Blast and find ZmFRO2,and the ZmFRO2 gene was cloned.To study the characteristics of ZmFRO2 gene and its protein,many works were done,such as bioinformatics analysis,expression pattern analysis and subcellular localization analysis.In order to analyze the physiological function of ZmFRO2,the ZmFRO2 overexpression vector was constructed and the genetically modified corn was aquired by using the method of agrobacterium-mediated infection.Then the related physiological indexes detection and phenotypic analysis were conducted on transgenic corn.The main results are as follows:1.In order to understand the characteristics of ZmFRO2 gene and its protein,bioinformatics analysis was carried out on ZmFRO2 gene and its protein.ZmFRO2 gene is located on chromosome 2,with a length of 4590bp and a cDNA length of 2585bp.ZmFRO2 protein is composed of 759 amino acids.After amino acid sequence alignment and prediction of transmembrane domain,it was found that ZmFRO2 protein has 10 transmembrane regions including the heme binding region with two pairs of histidine residues on the transmembrane region V and VII,and the reoxidation-reduction marker sequence in the transmembrane region VIII,which is highly conserved between the FROs.These results indicate that ZmFRO2 has the structural basis to play the role of iron reductase.2.In order to understand the influence of the expression and localization of ZmFRO2 on its function,the expression pattern and subcellular localization of ZmFRO2 in maize were analyzed.The results of Real-time RT-PCR relative expression analysis showed that under natural conditions in the field,ZmFRO2 was mainly expressed in leaves.It was speculated that ZmFRO2 mainly played the role of reducing iron in maize leaves.Otherwise,the expression of ZmFRO2 in the leaves and roots of seedlings increased after 24h and 6h Fe-deficiency treatment respectively,and then decreased,indicating that the roots were sensitive to iron deficiency.It was speculated that the expression of ZmFRO2 was first induced and then inhibited by iron deficiency.The subcellular localization results of protoplast in maize mesophyll showed that ZmFRO2 was located in the plasma membrane and cytoplasm.It was speculated that ZmFRO2 played a role in reducing iron on the plasma membrane and cytoplasm,and could reduce both extracellular and intracellular Fe3+.3.In order to study the effect of ZmFRO2-overexpression on iron absorption and transfer in maize,related physiological indexes of transgenic corn were determined.First of all,iron reductase activity of transgenic corn roots in Fe-deficiency condition was determinded by using the ultraviolet spectrophotometer.The results showed that after iron deficiency treatment 9 days,iron reductase activity increased significantly.It was speculated that ZmFRO2 has iron reductase activity.Next,AAS(Atomic Absorption Spectrometer)technique was developed for the determination of Zn/Fe content in parts of the transgenic corn growing in alkaline soil,the results showed that the Fe content of young root was 8.44?9.40 times more than the wild type,young leaves,mature leaves and grain were increased by 23.43%,42.53%and 17.11%.It was speculated that ZmFRO2 had the function of reducing Fe3+in roots and leaves,eventually increase grain Fe content.4.In order to figure out the effect of ZmFRO2-overexpression on agronomic traits of maize,the ZmFRO2 overexpression vector was constructed and the genetically modified corn was aquired by using the method of agrobacterium-mediated infection.According to the different test purposes,different ways of planting were executed.In the process of growth and after mature,the phenotype of transgenic corn was analyzed,and the results showed that the SPAD value of transgenic corn new leaves increased,the plant height reduced,panical weight and grains weight increased by 17.13%?44.35%and 18.07%?35.82%respectively than the wild type,hundred-grain weight increased significantly.It was speculated that overexpression of ZmFRO2 was able to increase maize yield by affecting agronomic traits,such as chlorophyll content and plant height.Based on the above results,it suggested that ZmFRO2 locates in the plasma membrane and cytoplasm and may participate in the iron reduction inside and outside the cell.ZmFRO2 mainly reduced iron in maize leaves,and its expression was firstly induced and then inhibited by iron deficiency condition.ZmFRO2 had the basic structure of iron reductase,the activity of iron reductase and the function of reducing iron.Overexpression of ZmFRO2 could increase the content of zinc and iron in maize plants and grains,and increase the yield of maize significantly.