Resistant-related Genes Screening And Analysis Of Defense Enzyme Of Maize Against Gray Leaf Spot

Gray leaf spot is a major leaf disease of maize in Northeast of China. The dynamicschanges of protective enzymes, the content of malonaldehyde (MDA) as well as the expressionsof disease resistance-related genes after the resistant/susceptible inbred lines (78599-1，OH43HtN/Ye478，K12) inoculated with Cercospora zeae-maydis strain were analyzed so that toanalyze the resistance physiological mechanism with the mutual reaction between maize andC. zeae-maydis. The resistant-related genes of maize against gray leaf spot were gained throughDDRT-PCR techniques，and molecular mechanisms of maize against gray leaf spot wereanalysed inicially. The main results are as follows:1. The gene expression difference among inbrid lines with different resistance before andafter inoculation with C. zeae-maydis were analysed using the technical systems of mRNAdifferential display and selecting sixteen pairs of random primers. After argumentation throughDDRT-PCR,46differential brands were obtained through polyacrylamide gel electrophoresisand silver staining detentions, and then40differential segments still expand out of the stripsafter collection and second argumentation with the collection rate up to87%. The resultsshowed that there is a mature mRNA differential display system in the screening of maizeagainst gray leaf spot.2. After reversed Northern hybridization detection,36positive segments obtained and thepositive rate is65%.19segments of T-Vector pMD19are successfully cloned, including5segments from Ye478,6segments from78599-1,5segments from ON43HtN and3segmentsfrom K12. There were six and five fragments respectively in resistant materials78599-1andOH43HtN，and the susceptible materials Ye478and K12have five and three fragments,respectively,the number of differentially expressed fragments of the latter is less than theformer.3. Sequence alignments of positive gene fragments obtained were analyzed about using the Blastsoftware in NCBI.The results showed that there were found the homologous fragments of Zeamays Mu tranposon insertion Mu1007701flanking sequence and Zea mays light-inducibleprotein CPRF-2in Ye478，OH43HtN.The homologous fragments of genes acylamino-acid-rel-easing enzyme have been found in Ye478and78599-1. There found the homologousfragments of MTN3gene,CONS TANS-CO5family, acyl-CoA-binding protein gene, Zea mayshypothetical protein just in Ye478, K12，OH43HtN，78599-1, respectively.The results showed that the resistant/susceptible inbred lines induced the different expressed genes induced geneexpression after inoculated with C. zeae-maydis, and the different roles played by the samegene.4. This study found that the changes in the activities of relevant defense enzyme in theinbred lines with different resistance become obvious after inoculation of C. zeae-maydis, theenzyme activities of PAL, POD and CAT obviously increase and higher than those of thesusceptible inbred lines, and the duration of the enzyme activity increase is longer than that ofthe susceptible inbred lines, indicating that the enzyme of POD, PAL and CAT play roles in thedisease process.The MDA contents of the resistant inbred lines are obviously lower than those ofthe susceptible inbred lines, indicating that the change of MDA contents is in negativecorrelation with the disease resistance degree.5. It is found that after inoculation with C. zeae-maydis, the CAT gene expression quantityof78599-1is higher than that of Ye478and K12, and the CAT expression quantity change inOH43HtN is not clear; However, the expression quantity of78599-1APX is obviously lowerthan that of OH43HtN, which indicates different signal paths of inbred lines with differentresistance in the defense against invasion of C. zeae-maydis.