Ultrastructural Changes And Cloning Of AvMYB10 And AvMYB12 Genes In Aquilegia Vulgaris Under Salt Stress

Aquilegia vulgars as an emerging domestic garden plant,has high ornamental value.In recent years,the area of saline land in China has shown an increasing trend,and the problem of soil salinization has become more serious,which affects the growth of ornamental plants and limits the application of ornamental plants.Therefore,it is important to carry out research on the mechanism of salt resistance in A.vulgars at the cellular and molecular levels.In this paper,we systematically investigated the ultrastructural characteristics of A.vulgars under Na Cl stress at different treatment times using transmission electron microscopy.Based on the transcriptomic data of pre-salt stress,we screened and cloned two genes,Av MYB10 and Av MYB12,from A.vulgars and performed bioinformatics analysis of the encoded proteins and gene expression patterns to provide basic information for further revealing the salt tolerance mechanism of plants.The results of the study are as follows:1.Under 200 mmol/L Na Cl stress,in the mesophyll cells,the increase in the number of mitochondria in A.vulgars may compensate for the reduced structure and function of individual internal cristae,and the proximity of mitochondria to chloroplasts allows the cell to efficiently use its limited energy to resist external salt stress.Changes in cystoid morphology affect photosynthesis in plants,and the increase in starvation granules may be due to the accumulation of cell membrane structures and pigment degradation products.The number of starch grains in both mesophyll cells and stem cells showed a trend of increasing and then decreasing with increasing treatment time,which can laterally reflect the operation of photosynthetic function in plants.In stem and root cells,the increase in vesicles reduced the adverse effects of contaminants released during organelle breakdown on plant cell structure and metabolic pathways,and the abundance of inclusions may be related to the high salt tolerance of A.vulgars.Stem cells underwent plasma wall separation after 9 d treatment compared with root cells after 6 d treatment,The cell walls were relatively more tolerant to salt stress,and the root cells showed more sensitivity to salt stress.After 9 d of Na Cl stress,the Golgi apparatus was degraded or even disappeared,which may be involved in cell wall and vesicle formation.2.All 68 R2R3-MYB transcription factors of A.vulgars contain R2 and R3 structural domains.Its protein amino acid number is between 189 and 512,molecular weight is between21624.08 and 56856.33 Da,isoelectric point is between 4.7 and 9.44,and protein fat coefficient is between 46.89 and 85.Predictions for subcellular localization indicate that all proteins are distributed in the nucleus and are hydrophilic.The protein sequences all contain three typical conserved elements,Motif 1,Motif 2 and Motif 3,and members of the same subgroup also have similar motif structures.By phylogenetic analysis,the R2R3-MYB transcription factors of A.vulgars were divided into 25 groups.Expression data analysis revealed that nine differentially expressed significant genes in A.vulgars were homologous to the known salt-resistant R2R3-MYB gene of Arabidopsis thaliana in response to salt stress.3.Two salt resistance-related genes,Av MYB10 and Av MYB12,were cloned from the leaves of A.vulgaris.Bioinformatics analysis revealed that Av MYB10 and Av MYB12,with ORFs of 639 bp and 990 bp,encoding 212 and 329 amino acids,respectively,are hydrophilic,unstable,non-transmembrane,non-secretory proteins.The secondary structures of Av MYB10 and Av MYB12 proteins showed mainly random coiling,while the tertiary structures of the proteins were formed by α-helix,β-folding and irregular coiling around each other.Av MYB10 has three glycosylation sites and 41 phosphorylation sites;Av MYB12 has four glycosylation sites and 54 phosphorylation sites.The results of constructing evolutionary tree analysis with other species with high homology showed that Aquilegia coerulea reached homology with Av MYB10 and Av MYB12 proteins,and Av MYB10 and Av MYB12 were closely related to the same buttercup family,Coptis chinensis and Anemonella thalictroides respectively.4.Fluorescence quantitative q RT-PCR results showed that Av MYB10 and Av MYB12 were differentially expressed in leaves and roots.Av MYB10 expression was significantly up-regulated in leaves,and the expression was significantly up-regulated and peaked after 12 hours of salt stress,followed by a decreasing trend;Av MYB12 expression was significantly up-regulated in roots,and the expression was significantly up-regulated and peaked after 48 hours of salt stress.These results suggest that Av MYB10 and Av MYB12 are differential genes in response to salt stress.