Cloning And Functional Study Of NtPOD_x Gene In Tobacco

Peroxidase?POD?plays a key role in the process of cellular reactive oxygen species?ROS?clearance.As an important economic crop and model crop in China,tobacco research on the molecular mechanism of tobacco NtPOD_x gene response to abiotic stress is of great significance.The main results obtained by NtPOD_x gene cloning,bioinformatics analysis,functional analysis and proteomics are as follows:?1?A POD gene belonging to type III secreted peroxidase was obtained from tobacco K326 by homologous cloning method.The full length of the sequence was 990 bp,encoding 329 amino acids,named NtPOD_x.Bioinformatics results showed that NtPOD_x protein It is a hydrophilic stable protein and also contains four binding sites and two conserved domains as one of the common features of the type III secreted peroxidase family;it has the highest homology with the flowering tobacco POD63,99%.The lowest homology with POD of Arabidopsis thaliana was 61%.The results of qRT-PCR showed that NtPOD_x gene was expressed in roots,stems,leaves and flowers.The gene was abiotic stresses such as salt,drought and low potassium.Induction;subcellular localization indicates that the NtPOD_x protein is localized in the cytoplasm.?2?After transferring NtPOD_x gene into tobacco K326,N1,N4 and N6 with higher expression of NtPOD_x transgenic tobacco were selected for abiotic stress test.The results showed that the transgenic type showed resistance to abiotic stress such as salt,drought and low potassium.Inverse.Functional studies of transgenic N1,N4 and N6 and wild-type K326 overexpressing the NtPOD_x gene showed that under salt stress,the transgenic genotype grew better than the wild type and accumulated less H₂O₂,MDA and Na⁺.Moreover,the content of K⁺,chlorophyll and soluble sugar was high,the photosynthetic index decreased little,and the activities of POD,CAT and SOD were high.The transgenic type increased the expression of K⁺transporter and antioxidant related genes.Under drought stress,the transgenic genotype grew better than the wild type,the photosynthetic index decreased less,the POD,CAT and SOD activities were high,the chlorophyll,soluble sugar and Pro content were higher,but the MDA content was lower;the transgenic type was higher than the wild type.Increased expression of antioxidant-related genes.Under low potassium stress,the transgenic genotype was longer than the wild type root and the K⁺accumulation was more.?3?Proteomic analysis of transgenic N1,N4,N6 and wild-type K326 tobacco under salt stress,and the results of differential protein were verified by qRT-PCR.The results showed that the transgenic line was 1.5 times more than the wild type.There are 123differential proteins,of which 89 are up-regulated and 34 are down-regulated;GO analysis indicates that cells and organelles are the main concentrated areas of these differential proteins,and their functions are mainly involved in catalytic activity and connectivity,and mainly regulate cell processes and metabolic processes.KEGG analysis showed that the differential proteins involved in the metabolic pathway were 1.5 times most,including:biosynthesis of secondary metabolites and carbon metabolism.