| Drought stress has become one of the most important factors affecting maize yield in Jilin province.Drought can affect the whole growth cycle of maize from seed germination,plant growth to reproductive growth.Drought caused the loss of maize yield.Therefore,it was significant to develop relevant molecular markers of drought-resistant related genes at seedling stage in maize.Molecular markers were used to assist the molecular breeding of maize and accelerate the breeding of drought-resistant maize inbred lines.In this study,68 maize elite germplasm resources were selected as association population.The drought-resistant candidate genes were mined by genome-wide association study and homologous gene cloning technology.The drought-resistant genes were identified by transgenic Arabidopsis.The association analysis of plant height and stem diameter at seedling and anthesis stage were carried out and the candidate genes were obtained.The main results are as follows:1.A total of 40,580 high quality markers were obtained by using gene chip technology to sequence the association population.The results of population structure analysis showed that association population was divided into five subgroups.The result of kinship analysis showed that the relationship between individuals was very far.The data of five drought resistance indexes including malondialdehyde content,superoxide dismutase activity,proline content,relative conductivity and leaf relative water content were analyzed.The results showed that the drought resistance degrees of association population were significantly different,the values were approximately normal distribution and the correlations between different drought resistance indexes were very weak.Tassel software was used for association population of five drought resistance indexes.A total of 8 significantly association markers were detected.The p values of these markers were 3.79×10-6-2.67×10-32 and the phenotypic contribution rates were 14.6-85.2%.Four markers were significantly association with the drought resistance index of malondialdehyde,three markers were significantly association with the drought resistance index of superoxide dismutase,and one marker was significantly association with the drought resistance index of relative conductivity.Five drought-resistant candidate genes were screened,there were Zm EREB160,Trehalose,MADS transcription factor,Calcium-dependent lipid-binding protein and Glutathione S-transferase.2.Bioinformatics and q RT-PCR analysis of Zm EREB160 gene showed that Zm EREB160 was a member of AP2/EREBP transcription factor and its expression levels were induced by drought,salt and ABA stress.Subcellular localization of Zm EREB160 was located in the nucleus and has transcriptional activation function in yeast.Zm EREB160 overexpression vectors were transformed into Arabidopsis,overexpressed plants were identified drought resistance at different growth stages.Zm EREB160 improved plant resistance to osmotic stress and ABA stress at seedling stage.The survival rates of overexpressed plants were higher than that of wild type plants under drought stress conditions.The survival rates of overexpressed plants were 60-79%,that of wild type plants was 30%,moreover,the expression levels of drought-resistant genes DREB2A,COR15A,RD29B and SAG13 in overexpressed plants were higher than those in wild type plants,and the expression levels of ABI2 and ABI5 genes in ABA signaling pathway in overexpressed plants were higher than those in wild type plants under drought stress conditions.The content of proline and malondialdehyde in overexpression plants were higher than that in wild type plants,and the activity of phenylalanine ammonia-lyase in overexpression plants was lower than that in wild type plants under drought stress conditions.The results showed that Zm EREB160 enhanced plant drought resistance by increasing the content of osmotic protective substances and expression of drought-resistant genes,and regulating the expression of genes in ABA signaling pathway.3.The results of ZmNAC33 gene analysis using bioinformatics and NAC genes evolutionary tree showed that ZmNAC33 was the homologous gene of ATAF1 and was a member of NAC transcription factor.The expression levels of ZmNAC33 gene were significantly induced by drought,salt and ABA stress.Subcellular localization of ZmNAC33 was located in the nucleus and has transcriptional activation function in yeast.ZmNAC33 overexpression vectors were transformed into Arabidopsis,overexpressed plants were identified drought resistance at different growth stages.ZmNAC33 improved seed germination rate of Arabidopsis under ABA and osmotic stress at seed germination stage.ZmNAC33 increased plant resistance to osmotic stress at seedling stage.The survival rates of overexpressed plants were higher than that of wild type plants under drought treatment.The survival rates of overexpressed plants were 62-81%,that of wild-type plants was 26.7%,moreover,the expression levels of drought-resistant genes DREB2A,COR47and RD29B in overexpressed plants were higher than those in wild type plants,and the expression levels of ABI5 genes in ABA signaling pathway in overexpressed plants were higher than those in wild type plants under drought stress conditions.The content of proline,malondialdehyde and the activity of superoxide dismutase in overexpression plants were higher than that in wild type plants under drought stress conditions.The results showed ZmNAC33 improved plant drought resistance by regulating expression of drought-resistant genes and participating in the regulation of genes in ABA signaling pathway.Combined with the results of evaluating drought resistance and ZmNAC33 gene sequences polymorphism of association population,haplotype Hap1 was identified as optimal haplotype.The results provided experimental basis for the development and utilization of functional markers in the future.4.Statistical analysis of plant height and stem diameter traits of association population at seedling and anthesis stages showed that the variation ranges of traits were wide and normal distribution.Tassel software was used for association population of plant height at seedling and anthesis stage.A total of 38 significantly association markers were detected by association analysis of plant height traits and a total of 16 significantly association markers by association analysis of stem diameter traits.Dwarf4 and RLD2 genes have been reported of controlling plant height in association markers at seedling stage.Six candidate genes of controlling plant height trait were also screened,such as transcription factor Zm LBD28,Zm GARS20,Zm GRAS70,Zmb HLH10,Zm OFP16 and Prenylated RAB acceptor.The results provided experimental data for further functional identification of candidate genes of controlling plant height trait. |
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