| Corn(Zea mays L.)is not only the most productive crop in the world,but also plays a very important role in industry and animal husbandry.With the complex changes in the current world situation and the steady,high-speed and healthy development of my country’s national economy and science and technology,the agricultural production mode have also undergone tremendous changes,and the breeding goals of maize have also changed.From the pursuit of high yield in the past to the pursuit of high yield,density resistance,lodging resistance,good quality,easy machine collection,etc.However,the long-term use of bicyclic breeding methods in our country,the germplasm resources of maize are narrow,and it is difficult to breed new varieties that meet the modern breeding goals.Therefore,the introduction of tropical and subtropical germplasm resources is one of the effective ways to solve the narrow problem of maize germplasm resources in our country.Germplasm resources in tropical and subtropical regions have strong photoperiodic responses.And photoperiod response sensitivity is the biggest obstacle to the utilization of exotic germplasm in temperate regions and blocks the expression of beneficial genes.By understanding the physiological,biochemical and genetic mechanisms of maize photoperiod,and the sensitivity to photoperiod is inactivated,which increases the possibility of breeding new varieties that meet the modern breeding goals.In our laboratory,the photoperiod-sensitive material CML288 and the photoperiod-insensitive material Huang Zaosi were treated under long-day light conditions(day: 15 h,dark: 9h),and the transcriptome analysis showed that the expression level of GRMZM2G171660(Zm ELF9)showed a very significant difference in the two materials.showed a very significant difference in CML288,And it was found that CML288 was 241 bp less than Huangzaosi at3446 bp upstream of the start codon,and there were many differences in SNPs were also found in the amplified promoter regions.The Zm ELF9 promoter activity was tested by transient dual luciferase assay.The analysis showed that the significant difference in the expression levels of Zm ELF9 between CML288 and Huangzaosi was caused by the 241-Indel in the promoter region.On this basis,the gene was cloned with the c DNAs of Huang Zaosi(HZ4)as templates to clone the gene,construct an overexpression vector,and carry out genetic transformation.After 3-4consecutive generations of selfing,a pure Zm ELF9-OX line was obtained.The phenotypic analysis of the homozygous Zm ELF9-OX line and the wild-type B104 plant under long-day conditions in Zhengzhou and short-day conditions in Hainan was carried out to further clarify the function of the candidate gene;subcellular localization and transcriptional activity analysis techniques were used to determine the Whether the gene has transcription factor characteristics;DAP-seq,gel electrophoresis migration assay(EMSA),and dual luciferase reporter system were used to further verify whether Zm ELF9 binds and regulates downstream target genes.This lays the foundation for further revealing the function and mechanism of Zm ELF9 gene in regulating maize flowering.The results of the study are as follows:1.Through phenotypic analysis,it was found that under long-day conditions,Zm ELF9 gene may negatively regulate maize flowering and plant height;under short-day conditions,Zm ELF9 gene has little effect on flowering time,but still negatively regulates maize plant height.2.By subcellular localization,Zm ELF9 protein was found to be expressed in the nucleus.The transcriptional activity analysis of Zm ELF9 by yeast transactivation system showed that the gene exhibited transcriptional activation activity;the transcriptional activity of this gene was further verified by Gal4-Lex A/UAS system,which was consistent with the results of yeast transactivation system.The experimental results indicated that Zm ELF9 may be a nuclear transcription factor.3.Through DAP-seq test and data analysis results,it was found that the binding motifs of Zm ELF9 with downstream target genes were preliminarily identified as: CGAAGSH,AGGGGGGAG and AACAWRT.It was further proved by EMSA that Zm ELF9 did bind to CGAAGGT and AACAAAT;further analysis of the binding peaks of the two biological replicates found that there were 18077 peaks in the two replicates,corresponding to 9373 genes.The intersection of the two repeats is located in 362 genes in the promoter region.Using GO enrichment analysis,it was found that these genes are mainly involved in m RNA splicing,regulation of meristem development,regulation of signal transduction,and the combination of NADP and NADPH.Combined with gene function annotation,16 target genes were selected that participate in photoperiod,circadian rhythm,flower development,etc.In order to preliminarily clarify the regulatory relationship between Zm ELF9 and these target genes,the dual-luciferase reporter assay was used to analyze.The results showed that Zm ELF9 inhibited the expression of Zm ZCN8,Zm ZCN16,Zm U2AF38,GRMZM2G151309,Zm DMT5 and Zm ADO1 genes,and preliminarily analyzed the regulatory network of Zm ELF9.In conclusion,Zm ELF9 may be a key gene regulating maize photoperiod sensitivity,and negatively regulate maize flowering,and it was preliminarily speculated that Zm ELF9 could delay maize flowering and affect maize photoperiod sensitivity by inhibiting the expression of Zm ZCN8,Zm ZCN16,Zm U2AF38,GRMZM2G151309,Zm DMT5 and Zm ADO1 genes. |
