Functional Analysis Of ZmMYC7 Gene In Maize

In nature,plants are subject to various stresses,including abiotic stresses(cold,heat,high salt,and drought),and biotic stresses(pathogen infection and mechanical damage to insects).In response to the stressful environment,the plants formed a systematic defense network after long-term evolution.The JA pathway is widely involved in the disease resistance of plants,deeply explore the key genes of the JA pathway and explore their molecular mechanisms of resistance that can provide a theoretical basis for the control and prevention of corn diseases.The laboratory acquires the maize ZmMYC7 gene by bioinformatics method,which is homologous to the Arabidopsis AtMYC2 gene.This study determined that the subcellular localization of the ZmMYC7 by the GFP fusion expression technique.By detecting the resistance of the maize B73 and the ZmMYC7 mutant plants to Fusarium graminearum and Setosphaeria turcica,it determined the function of ZmMYC7 during the maize resistance infestation.The interaction between ZmMYC7 and JAZ family genes in maize was studied by yeast two-hybrid technique(Y2H)and bimolecular fluorescence complementation(BiFC).Yeast one-hybrid technique(Y1H),it was used to identify,the downstream target gene regulated by ZmMYC7.The results lay the foundation for elucidating the molecular mechanism by which the ZmMYC7 gene participates in the JA pathway to produce disease resistance.The main results are as follows:1.Bioinformatics analysis of ZmMYC7.In this study,the ZmMYC7 gene(homologous to Arabidopsis AtMYC2)in maize was homologously analyzed,phylogenetic tree analysis,secondary(three)structure analysis,nuclear localization signal analysis,etc,it was found that ZmMYC7 has high homology with AtMYC2 and OsMYC2,ZmMYC7 has two domains: bHLH-MYC_N(basic helix-loop-helix-leucine zipper domain)and HLH,containing a nuclear localization signal: N-RPRKRGRKP-C,no signal peptide and transmembrane structure.2.Subcellular localization of ZmMYC7.The subcellular localization vector 103-ZmMYC7 of ZmMYC7 gene was constructed using the Gateway method;the transformation of 103-ZmMYC7 into tobacco leaves was carried out by Agrobacterium-mediated genetic transformation,and the GFP fluorescence signal was detected under a laser confocal microscope to confirm that the ZmMYC7 was localized in the nucleus.3.Identification of disease resistance of ZmMYC7 mutants.The EMS-mutated mutants of ZmMYC7(myc7-1 and myc7-2)were inoculated with Fusarium graminearum and Setosphaeria turcica.It was found that the ZmMYC7 mutants were more sensitive to Fusarium graminearum and Setosphaeria turcica than B73.The results indicated that ZmMYC7 played an important role in the corn resistance to Fusarium graminearum and Setosphaeria turcica.4.Analysis of disease resistance-related gene expression in ZmMYC7 mutants.The expression level of PR genes in the mutant was detected by Real-time PCR.The results showed that the expression levels of PR3,PR5,PR7 and PR10 in ZmMYC7 mutants were significantly lower than those in B73 in the late stage of infection;while the expression of PR1 in ZmMYC7 mutants was higher than that in B73;In the absence of pathogen infection(0 d),the expression levels of PR2 and PR4 in the mutants were significantly higher than in B73.When the pathogens infected for 1 d,the expression levels of PR2 and PR4 in the mutants were significantly lower than those of B73.It indicated that the mutation of ZmMYC7 affects the expression of maize PR genes,further indicating that ZmMYC7 plays an important role in the process of maize resistance.5.Screen for candidate interaction proteins of ZmMYC7.Using bioinformatics methods,phylogenetic analysis,conserved domain analysis,tissue-specific analysis,and the expression of maize JAZ family genes in resistance to biotic and abiotic stress were performed.Real-time PCR was used to detect maize inbred lines Mo17(resistant to Fusarium graminearum)and B73(susceptible Fusarium graminearum).The results showed that all of JAZ family members expressed tissue-specificity;under the biological stress(Aspergillus flavus and Fusarium verticillioides)and abiotic stress(high temperature,low temperature,high salt and ultraviolet damage),the expression level showed obvious changes;some JAZ family genes(such as ZmJAZ8、ZmJAZ20)showed significant differences in the expression of maize resistance to Fusarium graminearum inbred lines Mo17 and B73,and screened ZmJAZ8,ZmJAZ11,ZmJAZ12,ZmJAZ20 and ZmJAZ23 as candidate interaction proteins of ZmMYC7.6.Yeast two-hybrid(Y2H)identifies the interaction protein of ZmMYC7.The yeast two-hybrid technique was used to verification the interaction between ZmMYC7 and the candidate JAZ family members.As a result,compared with the negative control,the co-transformed AD-ZmMYC7+BD-ZmJAZ11,BD-ZmJAZ12 and BD-ZmMYC7+AD-ZmJAZ11,BD-ZmJAZ12 and BD-ZmMYC7+AD-AtJAZ1,AD-AtJAZ2,AD-AtJAZ3,AD-AtJAZ4,AD-AtJAZ5,AD-AtJAZ8,AD-AtJAZ9,AD-AtJAZ10,AD-AtJAZ12 yeast can grow normally on the three-deficient medium(-Leu/-Trp/-His)and the four-deficient medium(-Leu/-Trp/-His/-Ade),indicating ZmMYC7 and ZmJAZ11,ZmJAZ12,AtJAZ1,AtJAZ2,AtJAZ3,AtJAZ4,AtJAZ5,AtJAZ8,AtJAZ9,AtJAZ10 and AtJAZ12 can interact directly in yeast cells.7.Binary molecule fluorescence complementation of ZmMYC7 candidate interacting proteins.The interaction between ZmMYC7 and the maize JAZ family gene was verified by bimolecular fluorescence complementation.The results showed that fluorescent signals were present in tobacco leaves co-injected with nYFP-ZmMYC7 and cYFP-ZmJAZ11,cYFP-ZmJAZ12 and cYFP-ZmMYC7 combined with nYFP-ZmJAZ11 and nYFP-ZmJAZ12,but no fluorescent signal was detected in other negative controls.It shows that ZmMYC7 and ZmJAZ11,ZmJAZ12 can interact directly in tobacco leaves.8.Screening of ZmMYC7 candidate target genes and yeast one-hybrid identification.ZmERF147 and ZmLOX9 were screened as candidate target genes for ZmMYC7 by reviewing the literature.The expression of candidate target genes in ZmMYC7 mutant plants was analyzed by Real-time PCR,the expression level of ZmERF147 gene in ZmMYC7 mutants was found to be significantly lower than B73.Using the yeast one-hybrid technique to identify the target gene,it was found that the yeast co-transformed with AD-ZmMYC7 and pABAI-ZmERF147 grew well on the two-deficient + AbA medium compared with the negative control.ZmERF147 was shown to be a candidate target gene for ZmMYC7.