Screening Binding Proteins Of ZmDPS10-2 Promoter Related To Photoperiod Sensitivity In Maize

Maize photoperiod sensitivity is the main obstacle to tropical and subtropical germplasm in temperate use.The molecular mechanism of studying the key genes of photoperiod sensitivity in maize will provide theoretical basis and technical support for the effective utilization and improvement of tropical and subtropical maize germplasm.In this study,the main photoperiod-sensitive gene ZmDPS10-2 were selected as the test subject.The yeast one-hybrid library was constructed by using photoperiod-sensitive tropical inbred lines CML288,and the binding proteins of ZmDPS10-2 promoter was screened by yeast one-hybrid system.And to carry out bioinformatics function prediction analysis,so as to provide a basis for the analysis of the mechanism of action and regulatory research.The main findings are as follows:(1)Based on the study of photoperiod-sensitive gene ZmDPS10-2,the photoperiod-sensitive tropical maize inbred lines CML288 was used as experimental materials,we got the 1148bp of the core promoter fragment and the 756bp coding region fragment of the candidate gene,and the functional predictions were performed.It is found that this candidate gene is rich in light responsive elements,adversity and resistance elements,hormone regulatory elements,tissue-specific expression elements,and involved in drought,flavonoid biosynthesis and other resistance binding sites by prediction of promoter functional elements.It is presumed that the candidate gene is likely to be regulated by abiotic stress(drought,high temperature,etc.),and biological stress(disease,pest,etc.).The coding region and homology analysis showed that the candidate gene belonged to the NF-YC2 subunit transcription factor of the maize and had HMF conserved domain,which was highly similar to the HMF domain of Sorghum,Setaria italicagu and Oryza and so on,and have the close genetic relationship with Sorghum and Setaria italicagu.The results showed that ZmDPS10-2 candidate gene might play a role in the adversity regulation pathway of plant flowering,ER stress,heat stress and so on.(2)In this study,the total RNA was extracted from the leaves of the leaves of CML288 with different growth stages.The cDNA library was constructed by using SMART technology to synthesize cDNA.The quality of the library was evaluated based on the transformation efficiency and the length of the insert.It was found that the library capacity and titer were in accordance with the requirements.Most of the inserted fragments were between 750bp and 3000bp.On the whole,the quality of this cDNA library is good and can be used for subsequent experiments.(3)In this study,the candidate gene promoter region of 1180bp was cloned from the cDNA of CML288,and the decoy expression vector of yeast one-hybrid system was constructed.37 proteins were screened to interact with the target gene,and four proteins with related functional annotations were selected from them.The photoperiod and adversity related proteins Ribosomal L7Ae/L30e/S12e/Gadd45 family protein(Gadd45)and Ribosomal L14/L23 family protein(RPL23A),the endoplasmic reticulum response related protein bZIP transcription factor60(bZIP60),and the Adversity related proteins A member of Arabidopsis BAG(BAG),were constructed to the pGADT7 vector to do the further identification,and the interaction between them and candidate genes was confirmed.(4)The basic physical and chemical properties,secondary structure,hydrophobicity,signal peptide,transmembrane domain,subcellular localization and so on were predicted by bioinformatics.The results showed that the ribosomal protein Gadd45 and RPL23A may belong to the stable hydrophilic protein,easy to bind ligand,play a role in mitochondria;bZIP60 transcription factor and BAG protein may all belong to unstable hydrophilic protein,are also easy to bind with the ligand,which bZIP60 transcription factor may play a role in the cytoplasm,and BAG protein play a role in the nucleus.