Studies On The Transcriptome Of Maize Seedling Under Drought Stress And Characterizing CRISPR-Cas System In Microbes

Drought is one of the major factors limiting global maize production.Exposure to long-term drought conditions inhibits growth and leads to yield losses.Although several drought-responsive genes have been identified and functionally analyzed,the dynamic transcriptome in maize seedlings underlying responses to drought and water recovery treatments have not been fully elucidated.To characterize how maize seedling respond to drought stress at the transcriptional level,physiological responses and differentially expressed genes(DEGs)in the inbred line B73 under water deficit and recovery conditions were analysed.RNA sequencing analysis revealed that 6107 DEGs were responsive to drought stress and water recovery.Transcription factor genes from 37 families were differentially expressed among the three analyzed time-points.In total,61VQ genes were identified.The expression levels of most ZmVQ genes(57 out of 61 members)changed during the drought stress and water recovery.Quantitative real-time PCR analyses showed that most of the ZmVQ genes(41 out of 61 members)were responsive to NaCl treatment.Also,approximately half of the ZmVQ genes were co-expressed with ZmWRKY genes.Gene ontology enrichment analyses of the DEGs indicated that 50 GO terms,including those related to photosynthesis,carbohydrate metabolism,oxidoreductase activities,nutrient metabolism and other drought-responsive pathways,were over-represented in the drought-treated seedlings.The photosynthesis and cell wall development related genes were down-regulated in responses to drought stress.Additionally,our results showed that the content of gibberellin in drought treatment seedlings was decreased compared to that of control seedlings,while abscisic acid showed accumulated in the drought treated plants.The identified DEGs,especially those encoding transcription factors,represented potential targets for developing drought-tolerant maize lines.CRISPR-Cas systems in microbes,which defend foreign nucleic acids utilizing a single RNA-guided effector protein were analysed.By integrating microbes genomes and metagenome sequences,3426 non-rebundant Cas9 proteins were identified.Among them,1550 Cas9 were conserved based on known Cas9 which show cleavage activity in vivo and vitro.5 Cas9 with less amino acid were selected to further confirm its DNA cleavage in vivo and vitro.Additionally,we also identified two uncharacterized CRISPR-Cas systems which might be capable of target RNA cleavage,which encoded a single dual HEPN domain-containing large effector protein,cas13e and cas13f,respectively.Biochemical characterization revealed that Cas13e from Reichenbachiella agariperforans(Ra)and Cas13f from Microbial communities of pumphouse(Pu;metagenome)were capable of CRISPR RNA cleavage.However this target RNA cleavage in vivo and vitro need further verification.