| Soil salinization has a significant impact on the safe and healthy development of agriculture.Therefore,it is crucial to identify essential genes that respond to salt stress in plants and study the mechanisms underlying plant responses to such stress.Many studies have demonstrated the indispensability of WRKY-like transcription factors in plants under adverse environmental conditions.In this study,we investigated the activity of the NtWRKY65 promoter under salt and ABA stress,analyzed the transcriptional activity of NtWRKY65 using a yeast monohybrid method,explored the function of NtWRKY65 in salt stress through overexpression in tobacco material,and preliminarily investigated its role in salt stress by transcriptome analysis due to its significant increase under such conditions.The mechanism underlying the enhancement of salt tolerance by NtWRKY65 overexpression was investigated through transcriptome analysis,with the aim of identifying new genetic resources for molecular improvement of salt tolerance in tobacco varieties.The main findings are as follows:By constructing the NtWRKY65-Pro:GUS vector and transiently infiltrating tobacco seedlings under(150 mmol/L)Na Cl and(50 μM)ABA conditions,tobacco seedlings were found to exhibit stronger GUS staining under salt and ABA stress treatments,indicating that salt stress enhanced NtWRKY65 promoter activity;by constructing(p GBKT7-NtWRKY65)vector and yeast transformation,after 2 d of culture on SD/-Trp/-His/-Leu/-Ade/X-α-gal,it was found that the recombinant plasmid yeast strain of p GBKT7-NtWRKY65 grew blue spots in line with the positive yeast growth condition,which proved that the protein has transcriptional activation activity,thus supporting that the gene is a transcriptional factor.The function of NtWRKY65 overexpression lines(OE)and wild-type(WT)tobacco under salt stress was investigated using MS medium,hydroponic culture,and nutrient soil culture.The results indicated that the overexpressed lines(OE)significantly enhanced seed germination,seedling root length and survival rate,antioxidant enzyme activity,chlorophyll content,and photosynthetic performance.Meanwhile,they remarkably reduced malondialdehyde(MDA),hydrogen peroxide(H2O2),and conductivity content while increasing salt tolerance.The overexpression lines showed a significant increase in the relative expression of defense,antioxidant,salt,and ABA-responsive genes.In 10-week-old seedlings,the overexpression lines were involved in maintaining K+,Na+ ion homeostasis with 18.36%-20.43% and 14.26%-27.14% lower Na+ content in leaves and roots than wild-type plants respectively,as well as exhibiting 32.23%-49.16% and 16.31%-39.24%higher K+/Na+ ratios than wild-type plants in leaves and roots respectively.These findings suggest that NtWRKY65 expression under salt stress enhances tobacco seed germination and seedling survival,indicating a positive regulation of salt tolerance in tobacco plants by NtWRKY65.Furthermore,the upregulation of NtWRKY65 under salt stress improves the antioxidant capacity of tobacco plants,which is associated with ion homeostasis and defense-related gene expression.Transcriptome analysis of transgenic tobacco overexpressing NtWRKY65(OE5,OE11)and wild type plants under normal growth conditions and high salt stress revealed a total of4742 differentially expressed genes in response to high salt stress.The GO enrichment analysis revealed that the enriched pathways were associated with antioxidant activity,while KEGG metabolic pathway enrichment analysis indicated that phytohormone signaling,photosynthesis glutathione,starch and sucrose metabolism were the main enriched pathways.In addition,reactive oxygen species scavenging,phytohormone signaling,and Na+ and K+ion transport genes differed between WT and NtWRKY65 overexpression lines.It suggests that the salt tolerance mechanism of NtWRKY65 transcription factor response may be related to the reactive oxygen scavenging system,phytohormone signaling,and Na+,K+ ion transport pathway. |
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