Expression Analysis And Functional Study Of CePP2C19 Gene In Cyperus Esculentus Under Drought Stress

Cyperus esculentus is a kind of oil crop with economic value.Drought is not only an important factor affecting crop yield,but also restricts the yield of C.esculentus.The excavation and functional verification of drought response-related genes in C.esculentus can provide candidate gene resources for drought tolerance research.Protein phosphatase 2C(PP2Cs)is a type of phosphatase that is essential in drought-induced stress.It has been identified in many species,but its function and molecular mechanism have not been investigated.In this study,physiological indexes were detected and transcriptome sequencing were conducted on the samples of C.esculentus under drought stress.Bioinformatics and quantitative PCR techniques were used to extract the CePP2C19 gene of C.esculentus,and the gene was successfully cloned by molecular means to further explore its functional position and interaction with ABA receptors in ABA signaling pathway.It laid the foundation for elucidating the role of this gene in ABA signaling pathway.The function of CePP2C19 in drought and ABA stress response was identified by ectopic expression in yeast,Arabidopsis thaliana and Virus-induced gene silencing in C.esculentus.Overexpression of CePP2C19 gene could improve drought resistance by reducing ABA sensitivity.The main research results are as follows:1.Three concentrations of PEG6000 were set to simulate drought conditions,and the leaves of PEG6000 were wilted and curled after the concentration of 30% PEG6000.Therefore,the leaves and roots of PEG6000 under the stress were selected for the detection of physiological indicators.Combined with the analysis of physiological indicators,12 h was selected as the transcriptional sequencing samples.Based on transcriptome data analysis and the screening and verification of drought-related genes,the CePP2C19 gene was discovered.Bioinformatics analysis showed that 146 PP2 C family gene members existed in the full-length transcriptome of C.esculentus,which could be divided into 10 subfamilies according to Arabidopsis classification,and CePP2C19 belonged to subgroup A.2.The successful cloning of the CePP2C19 gene enabled subcellular localization analysis of the protein,which was then conducted using a tobacco transient transformation system.The results showed that CePP2C19 protein mainly functions in the nucleus.3.The potential interaction protein Ce PYR1 was screened through interaction network prediction and transcriptome data analysis,and the protein interaction was verified.The results of yeast two-hybrid experiment showed that CePP2C19 and Ce PYR1 could interact without ABA dependence,and Bi FC results further demonstrated that CePP2C19 and Ce PYR1 could interact in the nucleus.4.The drought resistance and ABA tolerance of CePP2C19 gene were preliminary identified by yeast system.The results of plate experiment of Arabidopsis thaliana showed that the germination rate of transgenic Arabidopsis thaliana was better than that of the wild type and the root length of transgenic Arabidopsis was longer than that of the wild type in the medium of drought stress and ABA stress.The phenotypes and physiological indexes of Arabidopsis thaliana in soil were analyzed under drought stress.The results indicated that transgenic strains were more drought tolerant than wild strains.The silencing of CePP2C19 gene in C.esculentus showed that the silencing of CePP2C19 gene in C.esculentus was more wilting than that in the control line under drought stress,and the ABA content of the silencing line was lower than that in the control line.The expression of Ce Sn RK2.3 gene in the ABA pathway was analyzed,and it was found that CePP2C19 gene had a regulatory effect on the downstream gene.These results indicated that CePP2C19 gene might be involved in the molecular regulation mechanism of drought resistance in C.esculentus.