The Cloning And Functional Analysis Of Antioxidant Related Gene Zm-APX In Maize

Maize(Zea mays L.)have important significance to food security and economic development as an important forage crop,food crop and industrial raw material.Plant face multiple environmental challenges at the same time in their whole life,including biotic(viruses,insects,etc.)and abiotic(drought,high temperature,salt)stress,which can cause serious influence on maize growth,resulting in the decrease of maize yield or even total loss.Corn is not like animals can moving their locations or by macrophages in the body to avoid the damage,but the corn has its unique defense mechanism,how to discover and separation the important proteins in defense reaction and analysis these proteins in vitro,which have important theoretical and practical significance in exploring maize defense reaction.In this study,we use maize inbred lines B73 as experimental material,whose genome has been sequenced,on the basis of the proteomics research Sugarcane Mosaic virus(SCMV)infection resistant genotype Si Yi and susceptible genotype B73,a one differentially up expressed protein ascorbate peroxidase(APX)is studied,the main research results are as following:1、 Using homology-based cloning technology,the Zm-APX gene was cloned.we obtain a 1186 bp c DAN sequence,which contains a 750 bp coding box;the ORF encoding a 250 amino acids protein,which molecular weight is 27.4 k D,PI is 5.64;The ORF and its corresponding DNA sequence alignment analysis found that: the DNA sequence contains nine exons and eight introns.Zm-APX does not exist transmembrane domain and signal peptide,suggesting that the protein may belong to intracellular protein,located in the cytoplasm;APX homology comparison showed that Zm-APX and sorghum APX have a near relationship,followed by corn and rice,cotton APX is relatively far;Zm-APX gene express in various organizations in maize.2、To study the Zm-APX gene expression patterns,we determine the relative expression of Zm – APX in B73 seedlings under abiotic(drought,high temperature,high salt,hormones)stress by using the real-time fluorescent PCR(RT-PCR)technique.Results showed that under the high temperature(370 C)processing,the relative expression of Zm-APX rise rapidly,reached peak after processing for 3 h,followed by decreasing,but still remain a level above the control;Under drought which keep the normal physiological condition treatment,along with the severe drought degree,Zm-APX relative expression increasing;Under 350 m M Na Cl treatment,the relative expression of Zm-APX reached peak at 12 h after treated,about three times compare with control;The expression patterns different under the hormone(ABA,SA)treatments,under ABA treatment,the relative expression of Zm-APX always maintain about three times compared to control,while under SA treatment,Zm-APX relative expression showed increase first and followed by decrease,take all above in conclusion,we can speculate that Zm-APX is closely related to the abiotic stress resistance in maize.3、We constructed the fluorescent expression vector p CAMBIA1304-Zm-APX,by tobacco instantaneous expression experiments mediated by agrobacterium tumefaciens,observe the expression of green fluorescent distribution under confocal microscope,confirm that the localization of Zm-APX protein is a cytoplasm protein.We constructed the expression vector p CAMBIA3301-Zm-APX,through transfixion Arabidopsis by tidbits infection method,selecting stable expression Arabidopsis thaliana plants.Under abiotic stresses(drought,high temperature and high salt),p CAMBIA3301-Zm-APX Arabidopsis compared with p CAMBIA3301 Arabidopsis and wild-type arabidopsis,after a week of drought the three genotypes Arabidopsis all with death phenomenon,but the survival rate were 32%,5% and 1% respectively,after watered for a week,the survival rates were 80%,8% and 80% respectively;after a week of high temperature treatment,survival rates were 75%,25% and25% respectively;High salt process for a week,the survival rate were78%,12% and 11% respectively,these results suggesting that Zm-APX gene has a positive regulation in enhancing of Arabidopsis resistance to abiotic stress,but has a stronger resistance to drought and high salt,it may be associated with the specific role of APX isozymes.In the abiotic stresses(drought,high temperature and high salt)processing,p CAMBIA3301-Zm-APX Arabidopsis thaliana,ABA1 and ABA2 relative expression level both have an significant up regulation,ABA1 and ABA2 are key genes in regulating of plant hormone ABA synthesis,we can inferring that the ability of Zm-APX in enhancing the Arabidopsis thaliana resistance to abiotic stress is closely related to the synthesis of ABA.