| Water stress affected the growth,development,yield and germplasm of plants seriously.The molecular mechanisms of plants responsing to drought stress include pre-transcriptional epigenetic regulation and the participation of signal molecules such as post-transcriptional hormones,transcription factors and stress response genes.DNA methylation is the earliest and most deeply studied epigenetic modification.When plants are subjected to drought stress,they will make a response through DNA methylation to help them cope with drought stress quickly.Water loss of potato during tuber formation and expansion seriously affected the normal growth and development of potato plants.There are few studies on the role of DNA methylation in regulating the epigenetics of potato under drought stress.Therefore,in this paper,five potato varieties with different drought resistance were used as experimental materials to analyze their phenotypic and physiological characteristics under mannitol simulated drought stress,DNA methylation inhibitor treatment and co-treatment,and then transcriptome analysis was carried out in two potato varieties with extreme response to drought and DNA methylation inhibitors.Finally,the StGST gene family regulated by DNA methylation in response to drought stress was identified and analyzed.The results are as follows:1.Five potato cultivars Atlantic,Favorita,CIP 398098.119,CIP397077.16 and Qingshu 9 were used as experimental materials;the plantlets of five cultivars were incubated with the regular MS medium,the MS medium supplemented with 60?mol L-1methylation inhibitor?5-aza-2?-deoxycytidine?,200 mmol L-1 mannitol,and 60?mol L-1methylation inhibitor combined with 200 mmol L-1 mannitol for 24 days;then phenotypic traits including both physiological and biochemical parameters were collected and comprehensively analyzed.The results showed that the response of different varieties to mannitol and 5-azadC was similar,the response of Qingshu 9 indicates this variety is least sensitive to mannitol and 5-azadC among others,while Atlantic is the most sensitive variety.The effects of drought stress and DNA methylation inhibitor on the growth of potato test-tube plantlets were compared.Dry and fresh weight,aboveground height,leaf number and chlorophyll content decreased significantly?P<0.05?,SOD,POD and CAT activities increased significantly?P<0.05?,proline and MDA content increased significantly?P<0.05?,but branch number,root length and average root diameter did not change significantly.These results suggest that under the stressed condition,the different traits might have different regulatory pathways include DNA methylation.the phenotypic traits of plantlets from the combined treatment of drought and 5-azadC were comparatively analyzed against single treatment,the data indicates that dual-treatment further inhibited the growth,and included strong stress response such as high activities of SOD,POD,CAT activity and increased MDA and Pro content.the results from this study suggest that the DNA methylation might involve in potato in response to drought stress,even regulatory capacity various among genotypes.This will provide a preliminary data for further study on epigenetic regulatory mechanism of potato in response to drought and other abiotic stresses,and also will open a new practical direction of potato improvement in the genomic editing era.2.The transcriptional analysis of two potato varieties with different drought resistance under mannitol simulated drought and 5-azadC treatment was carried out,and the differentially expressed genes?DEG?were screened by Fold-change>2 and corrected P<0.01.The results showed that the two potato varieties with different drought resistance were treated with mannitol and mannitol.GO enrichment analysis showed that both treatments were co-enriched to GO term related to oxidative stress and carbohydrate metabolism.The results showed that these GO term-related genes were regulated by DNA demethylation in response to drought stress in different drought-tolerant potatoes.At the same time,1345 DEGS responding to drought and DNA demethylation were enriched by KEGG function.The results showed that plant drought resistance related pathways were plant MAPK signal pathway,plant glutathione metabolism pathway,glycolysis and gluconeogenesis,phosphate inositol metabolism pathway and plant hormone signal transduction pathway.The results showed that the difference of response of these pathway-related genes to drought in Atlantic and Qingshu 9 potato varieties was regulated by DNA methylation.At the same time,the 1500 bp study on the DEGs promoter region showed that the GST gene involved in plant glutathione metabolism under drought stress reduced the methylation level of ABRE and CAAT-box acting elements in the promoter region through DNA demethylation,and then activated the expression of the gene to cope with drought stress.3.Based on the results of transcriptome analysis,the whole gene family analysis of potato glutathione S-transferase showed that 70 potato GST genes were identified in the whole potato genome.these proteins were divided into 9 different subclasses by domain analysis,multiple sequence alignment and phylogenetic analysis.At the same time,like other plant species studying the GST family,Tau is also the largest subclass of potato GST genes,with 53 members.The comparative genomic collinearity maps of potato,Arabidopsis thaliana,pepper and tomato were also compared.33 potato GST genes showed homology with tomato GST genes,followed by pepper?17?and Arabidopsis thaliana?11?.Some potato GST genes have been found to be associated with at least two pairs of homologous genes?especially between potato and Arabidopsis thaliana and pepper GST genes?.Such as StGSTU36 and StGSTU42,indicate that these genes may play a major role in the evolution of plant GST gene family.1500 bp analysis of the promoter region of StGST gene showed that there were ABRE and CAAT-box acting elements related to plant response to drought stress in the CpG island region of gene StGSTT1,and MYB,CAAT-box and MYC cis regulatory elements related to plant response to drought stress in the CpG island region of StGSTU14 gene,indicating that potato regulates the methylation level of ABRE,MYB,CAAT-box and MYC acting elements in response to drought stress.And then regulate the expression of genes to cope with drought stress.To sum up,this study not only provides a preliminary theoretical basis and experimental ideas for in-depth study of the epigenetic regulatory network pathway involved in the regulation of potato drought stress response by DNA methylation,but also provides a theoretical basis for opening up a new method of potato molecular breeding for drought resistance.However,the mechanism of DNA methylation regulating potato response to drought stress is not clear.In the future,we can further analyze its biological significance by detecting the level of DNA methylation after drought stress. |
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