Function And Molecular Mechanism Analysis Of ZmRL6 Responsive For Drought Tolerance During Seeding Stage In Maize(Zea May L.)

MYB-related gene is a subclass of MYB transcription factors,which mainly participates in the processes of secondary metabolism,growth and development,biological and abiotic stress.However,only few reports on the role of MYB-related transcription factors in response to drought stress and regulatory mechanism in maize.In this study,the differentially expressed MYB-related transcription factors identified from the former transcriptome results under drought-rewatering treatment were systematically analyzed,and ZmRL6,strongly responded to drought stress,was selected as the candidate gene.Cloning and functional verification,interaction protein screening and identifying directly target genes were conducted,and the results preliminary analyzed the molecular mechanism of ZmRL6 in response to drought stress in maize seedling stage.The main results were as follows:1.Systemic analysis was conducted for 46 maize MYB-related genes screened from the former transcriptome under drought and rewatering treatments.The results showed that 46 genes were unevenly distributed on 8 chromosomes and divided into six sub-groups by phylogenetic analysis.The mainly stress-related cis-elements ABRE,MBS,ARE,LTR and hormone response element TCA-element?TGACG-motif?TGA-element were identified in the promoter regions of MYBrelated genes.In addition,9 core genes were identified by co-expression network analysis.Among them,ZmRL6 was strongly responsive to drought stress.2.Sequence analysis showed that the ORF of ZmRL6 was 291 bp,encoded 96 amino acids,and the molecular weight was 10.7k Da,isoelectric point was 8.14;Sequence alignment revealed that ZmRL6 MYB-related shared high sequence homology with Setaria italica and Hordeum vulgare.qRT-PCR analysis showed that ZmRL6 had the highest expression in stem,and ZmRL6 could be induced by drought,salt and heat,respectively.Also,ZmRL6 was up-regulated under different hormone stresses.Subcellular localization revealed that ZmRL6 protein was nuclear protein.3.The overexpression and CRISPR/Cas9 knockout vectors of ZmRL6 were constructed and transformed into maize inbred line B104 by Agrobacterium-mediated method,and obtained ZmRL6-OE and ZmRL6-Mut transgenic plants.Seedings of T3 transgenic lines and WT plants stopped watering to practice natural drought stress for 10 days.The results showed that under drought treatment,maize seedlings grew slowly.Compared with WT plants,the leaves of ZmRL6-OE plants significantly delayed leaf rolling and no curling phenomenon.The malondialdehyde(MDA)content and relative electrolyte leakage(REL)of ZmRL6-OE plants were lower than that WT plants,and the peroxidase(POD)activity,superoxide dismutase(SOD)activity and proline(Pro)content of ZmRL6-OE plants were higher than that WT plants.While ZmRL6-Mut plants exhibited severe leaf wilting,and the leaves curled and the tips turned yellow.ZmRL6-Mut plants showed increased drought sensitivity evaluated by higher MDA content and relative electrolyte leakage(REL),lower peroxidase(POD)activity,superoxide dismutase(SOD)activity and proline(Pro)content than that in WT plants.These results indicated that ZmRL6 positively enhanced tolerance to drought stress by eliminating ROS,inhibiting cell membrane damage and cell death.4.20 candidate proteins which may interact with ZmRL6 protein were screened from the yeast library by yeast two-hybrid technology,ZmRL6 protein as bait.Three interacting proteins were verified,they were SNF1-related protein kinase catalytic(Zm00001d005108,SnRK1a),Malate dehydrogenase(Zm00001d034241,MDH)and Chaperone protein Dna J(Zm00001d024635,Dna J).These interactions might be involved in ABA signal transduction,cell signal transduction pathways,response to oxidative stress,chloroplast biosynthesis to increase drought tolerance in plants.5.8 target genes of ZmRL6 were identified by RNA-seq and DAP-seq,which mainly involved in phytohormone signal transduction(Zm LAX3 and Zm GASA13),sugar metabolism(Zm IRX7 and Zm UGT88A1),lignin synthesis(Zm MYB4 and Zm MYB6)and antioxidative stress(Zm PRX Q and Zm CYP71B3).RNA-seq results showed that the expression levels of Zm CYP71B3,Zm PRX Q and Zm LAX3 in ZmRL6-OE plants were higher than that in WT plants,and the expression levels of Zm CYP71B3,Zm PRX Q and Zm LAX3 in ZmRL6-Mut plants were lower than that in WT plants.The expressions of Zm GASA13,Zm MYB6,Zm MYB4,Zm IRX7 and Zm UGT88A1 showed the opposite trend.The double luciferase assay showed that ZmRL6 inhibited the promoters of Zm GASA13,Zm UGT88A1,Zm IRX7,Zm MYB4 and Zm MYB6,and promoted the promoters of genes Zm LAX3,Zm PRX Q and Zm CYP71B3,which was consistent with the results of RNA-seq.These results suggested that ZmRL6 may enhance tolerance to drought stress by participating in oxidative stress,lignin synthesis,sugar metabolism,phytohormone signal transduction pathways.