Phenotype Identification And Protein Analysis Of Tobacco Leaf Vein Fusion Mutants

Tobacco(Nicotiana tobacum L.)is an important economic crop in China.Among the many mutants of tobacco,leaf vein mutants are relatively rare.Mutation of tobacco veins will change the leaf morphology of tobacco plants,affecting the growth and development,yield and quality of tobacco.This experiment carried out related research on tobacco leaf vein mutants found in the field,through dynamic observation of leaf development,observation of leaf veins and leaf structure,analysis of proteome expression,determination of common physiological indicators and quantitative analysis of genes corresponding to related differentially expressed proteins.Initially explore its physiological and biochemical index changes and molecular mechanism of leaf vein mutation to understand the related information of this leaf vein mutant.The research results are as follows:1.Phenotype observation of tobacco leaf vein mutants(1)Leaf vein leaf shape observation.Mutant plants and wild-type tobacco seedlings with the same growth status were selected,and the leaf phenotypic traits of the two stages were observed at the small cross stage,the large cross stage,the four-leaf stage,and the eight-leaf stage.The traits of the mutants were basically stable when they developed to the eight-leaf stage,showing that the veins were fused and there was no obvious distinguishing feature between the main veins and the lateral veins.The lateral veins and the main veins at all levels began to differentiate and grow in the leaf primordia and the veins were curved.Completely,the surface of the blade appears convex and concave,and the leaf shape is irregular semi-circular or fan-shaped.The phenotype of wild-type leaves is normal.(2)Observation of paraffin sections of veins.The leaf veins of the wild-type and mutant strains at the four-leaf stage and the eight-leaf stage were selected for paraffin sectioning to observe the differences in the vascular bundles of the leaf veins.The vascular tissue of the mutant veins was loosely arranged,and the eight-leaf stage was more obvious than the four-leaf stage.The vascular tissue of the wild-type leaf veins is neatly arranged,and the lateral veins on the dorsal axis are blurred and loosely arranged.(3)Scanning electron microscopy of the leaves.Under the same field of view(100um),the mutants have more stomata than the wild type,with 6.3 in the wild type and 8.3 in the mutant;the stomata of the mutant are larger than in the wild type;there are differences in leaf epidermal cells,and the leaf epidermal cell boundaries Blurred,changeable morphology,more impurities attached to the surface,clear boundaries of wild-type leaf epidermal cells,consistent morphology,smooth and clean surface.(4)Observation of leaf submicroscopic structure.There is no significant difference in the cell wall thickness between the mutant and wild-type vascular bundle sheath cells.The morphology of the mutant is irregular and the wild-type is oval-like.The mitochondrial swelling of the leaf of the mutant is more than that of the wild Mild and the accumulation of starch granules in the chloroplast is less than the wild type.2.Determination of physiological indexes of tobacco leaf vein mutantsThe following conclusions were drawn from the determination of the relevant physiological indexes of the tobacco plant at the eight-leaf stage: the proline,MDA,soluble protein and soluble sugar in the wild-type and mutant strains all increased with time during the three sampling stages,but The increments were significantly greater than that of wild type;the biomass of wild type and mutant strains increased with time.The wild type had more weight than the mutant strain,but the dry matter rate of the mutant strain was greater than that of the wild type;The photosynthetic pigment content of the mutant strain and the mutant strain increased with time but the mutant strain was higher than the wild type.All photosynthetic performance indicators increased with time,and mutants were higher than wild type.The SOD,POD,CAT enzyme activity and total antioxidant capacity of the mutants were higher than those of the wild type.The content of IAA and CTK at the base of leaves of wild-type and mutant strains are higher than those at the middle and tip of leaves;the content of IAA at the tip of leaves increases with time and the difference becomes more and more significant.The content of the leaf tip of the plant has an increasing trend,but there is no such trend in the wild type.3.iTRAQ analysis of differentially expressed proteins in tobacco leaf vein mutantsThe wild-type and mutant strains at the eight-leaf stage were selected for proteome determination.The total number of detections by iTRAQ analysis was 232,400,and the number of matched spectra was 44,184,accounting for 19.0%.The number of identified peptides(Peptides)was 21878,and the number of unique peptide sequences(UniquePeptide)was 14645.The three samples S1,S2,S3 and wild-type octaphyllum taken at the eight-leaf stage of the mutant The number of identified proteins(Identified proteins)of the three samples W1,W2 and W3 taken during the period was 5593.The number of quantifiable proteins is 4491.There are a total of 221 proteins with S / W Ratio greater than 1.5 or less than 1 / 1.5,39 up-regulated proteins and 182 down-regulated proteins through GO annotation,subcellular structure annotation,KEGG enrichment analysis,protein interaction analysis,Screened two proteins with obvious differential expression and related to plant roots,stems and leaves development A0A1S4CRZ6 and P22091,the former is involved in the composition of the ribosomal protein 60 S large subunit L23 The latter is mainly composed of chitinase.Real-time fluorescence quantitative analysis of their corresponding genes LOC107821921 and CHN50.4.Analysis of relative expression of genes CHN50 and LOC107821921 in mutants and wild typeComparing the relative expression levels of the genes CHN50 and LOC107821921 in the roots,stems and leaves of the plants,it can be found that the relative expression levels of the two genes in the stems are the smallest,and the relative expression levels in the roots are second highest in the leaves Moreover,the expression level of CHN50 in the mutant strain was smaller than that in the wild type,and the relative expression level of LOC107821921 in the wild type plant was smaller than that in the mutant strain,which was in agreement with the protein detection results.