Screening And Verification Of Upstream Regulators Of Photosynthetic Key Enzyme Gene ZmRbcS1 In Maize

Droughts are the primary limiting factor for maize(Zea mays L)production.Photosynthesis is highly sensitive to drought stress.As a key enzyme in photosynthetic carbon assimilation,Rubisco small subunits(RbcS)of Rubisco affect the function of Rubisco holoenzyme.The protein ZmRbcS1 encoding the small subunit of Rubisco was significantly upregulated from the maize proteome in response to drought stress in the early stage of the laboratory,suggesting that this protein plays a positive regulatory role in maize response to drought stress.This test by maize inbred line B73,ZmRbcS1 mutant(Mu),ZmRbcS1 genes for the initial functional verification,and connecting with the bioinfonnatics analysis of the promoter cis-element,using yeast single hybridization technology screening ZmRbcS1 upstream promoter transcriptional regulatory factor,and select two transcription factors made to cover further validation.The main research results are as follows:1.Subcellular localization and expression pattern analysis of ZmRbcS1For B73 maize leaf cDNA template,this study design primers amplification ZmRbcS1 open reading code box(ORF)gene,encoding 170 amino acids,after built into the plant expression vector with GFP label pGWB605,via agrobacterium infect this type of tobacco leaf,the laser confocal microscope found that the gene encoding protein product positioning in the chloroplast.RNA was extracted from the roots,stems,leaves,female panicles and male panicles of B73 plants and cDNA was obtained by reverse transcriptional reaction.The results of fluorescence quantitative test showed that the main expression sites of the gene were leaves,followed by male panicles(54.7%of leaves),stem(34.2%of leaves)and female panicles(9.3%of leaves),but no expression was found in roots.2.P henotype and resistance identification of ZmRbcS1 mutantCompared with wild-type B73,ZmRbcS1 mutant(Mu)decreased leaf area by 76.3%,shoot fresh weight by 82.6%,and root fresh weight by 88.8%.Under drought stress,Mu leaves suffered severe water loss and wilting,and the expression level of RbcS1 gene in Mu leaves decreased by 70.8%compared with normal conditions.The results showed that the content of Chlorophyll A(Ca),Chlorophyll B(Cb)and Chlorophyll content(CT)in leaves of Mu were lower than that of B73 under normal and dry conditions.The results of antioxidant enzymes and membrane lipid peroxides showed that the SOD activity of Mu leaves was lower than that of B73 leaves under both normal and drought stress.POD activity increased slightly,and the activity of Mu was higher than that of B73.After drought stress,MDA in Mu leaves increased by 47.8%compared with normal conditions.3.Analysis of cis-acting elements of ZmRbcS1 promoterBy analyzing the cis-acting elements of the ZmRbcS1 gene promoter,we found that the cis-acting elements of the plant core promoter were CACA-box and TATA-box.Light response elements ATCT-motif,G-box,GATA-motif,GT1-motif,I-box，Sp1,TCT-motif,RbcS-CMA7C;Plant hormone response elements ABRE,CGTCA-motif,P-box,TGACG-motif,and elements ARE,MBS,etc.in response to environmental stress.The discovery of these acting elements suggested that ZmRbcS1 gene expression might be induced by stress conditions.4.Single hybrid screening of transcription factors and validation of interactions in yeastThe bait plasmid and library plasmid were transfected into Y1H GOLD.A total of 6 transcription factors(NAC120,G2-like7,MYB-related40,GEBP17,GNAT41,NTF2)and 60 proteins were screened by monoclonal positive detection,sequencing and sequence alignment under the ABA concentration of 400 ng/mL.Fluorescence quantitative analysis of transcription factors under drought stress revealed that only MYB-related40 was up-regulated after 12 h of drought stress,while the rest were down-regulated.Further transcriptional activation activities of two transcription factors,NAC120 and G2-like7,were detected,and it was found that both ZmNAC120 and ZmG2-like7 had transcriptional activation activities.The yeast single hybrid rotation test showed that both ZmNAC120 and ZmG2-like7 could interact with ZmRbcS1 in yeast,and the dual luciferase reporter gene system test results showed that ZmNAC120 significantly increased the promoter actiyity of ZmRbcS1.