| Maize is one of the three major food crops in the world,and abundant maize germplasm resources are the basis for the long-term and stable development of maize research.Tropical and subtropical maize has a rich genetic variation basis,so it has become an important source of rich maize germplasm resources.Photoperiod sensitivity is the main obstacle to the utilization of tropical and subtropical germplasm in temperate regions.Therefore,it is of great significance to study the regulation mechanism of key genes of maize photoperiod sensitivity for the improvement of maize germplasm.The research group used the tropical maize inbred line CML288 in the early stage to obtain the related gene ZmGI that regulates the photoperiod pathway through homologous cloning,and carried out functional analysis of ZmGI.On this basis,this study identified the regulatory binding sites of ZmGI by DAP-seq technology,and analyzed the genes bound to it.The transcriptional activity analysis of the upstream genes of ZmGI and the dual luciferase reporter system test proved that The regulatory relationship between upstream transcription factors and ZmGI,and the interaction network of ZmGI was initially constructed.The main findings are as follows:1.Using protoplast transformation technology,the subcellular localization of ZmGI gene,the results of subcellular localization showed that ZmGI was expressed in both cell membrane and nucleus,but it was mainly located in the nucleus.2.The Transcriptional activity of ZmGI was analyzed by yeast transactivation system and Gal4-Lex A/UAS system.In the yeast system and the Gal4-Lex A/UAS system,it showed the same transcriptional activation activity,indicating that the gene ZmGI has transcriptional activation activity and plays an activating effect on downstream genes.3.Using DAP-seq technology,the B73 inbred line was used to construct a DNA library,and the downstream target genes of ZmGI were screened.Using MEME software to analyze the data obtained by DAP-seq technology,it was preliminarily confirmed that the binding sites of ZmGI and target genes were CCGAGG,CACCGGAC and ACGGGGAG.Further analysis found that there were 8305 target genes in common between the two biological repeats,of which 1355 target genes acted on the promoter region.GO enrichment analysis was performed on the target genes acting on the promoter region,and the results showed that these genes were mainly involved in cellular components,molecular functions and biological processes.It is involved in photoperiod pathway,regulation of plant flowering time,photomorphogenesis,and response to stress.Through functional annotation of downstream target genes,26 genes were found that may be relate to flower time.Five genes were randomly selected,and the effect of ZmGI on the promoter activity of these target genes was analyzed by the dual-luciferase reporter system assay.The results showed that ZmGI was involved in the regulation of downstream target genes such as E2 11(Zm00001d012513),XBAT31(Zm00001d028254),Zm RUB(Zm00001d018190),DHN1(Zm00001d037894),SRK2A(Zm00001d003758).4.Three transcription factors acting on the ZmGI promoter region were obtained by ATAC-seq in the early stage:HSFA6b1,HSFA6b2(heat stress and plant flowering),and SPL(reproductive system development and flowering)using the dual luciferase system test.The effect of HSFA6b1,HSA6B2 and SPL on the promoter activity of the downstream target gene ZmGI was analyzed.The results showed that the transcription factors HSFA6b1,HSA6B2 and SPL all promoted the ZmGI promoter activity.In summary,ZmGI as a key regulator of photoperiod flowering,is involved in the regulation of downstream photoperiod genes as a transcription factor to regulate the flowering time of maize.At the same time,ZmGI can also participate in the regulation of plant flowering through other regulatory pathways.In addition,ZmGI is also involved in the regulation of organ development. |
