| To investigate the phenotypic differences of salt-alkali tolerant rice germplasm and to identify new genes regulating salt-alkali tolerance and analyze the molecular regulatory mechanisms, this study used 284 rice germplasm resources collected and developed in the laboratory as materials. A 50 mM Na2CO3 hydroponic stress system was established, combined with potted salt-alkali soil (pH 9.5) stress verification. Key indicators such as seed germination, seedling morphology, antioxidant enzyme (SOD, POD, CAT) activities, and yield components were measured, and two highly salt-alkali tolerant materials (866 and 2069) and two salt-sensitive materials (886 and 865) were selected. Potted stress treatments were carried out in mixed salt-alkali soil (SA) with conventional soil at pH 7.0 as control (CK). Materials 2069 and 865 were selected for sequencing, and root and leaf tissues at the five-leaf stage were used for transcriptome sequencing. Differentially expressed gene analysis, GO functional enrichment, and KEGG pathway enrichment analyses were conducted, and candidate gene expression levels were validated by qRT-PCR. The results showed that 4,465 differentially expressed genes were identified. Ion homeostasis and antioxidant defense pathways were the core pathways for salt-alkali tolerance. GO enrichment analysis indicated that the differential genes were mainly involved in biological processes such as ion transmembrane transport, reactive oxygen species clearance, and stress signal transduction. KEGG enrichment analysis revealed significant enrichment in glutathione metabolism, flavonoid biosynthesis, starch and sucrose metabolism, and Ca2+ signaling pathways. Further validation of gene transcription levels by qRT-PCR identified 10 candidate salt-alkali tolerance genes that were significantly upregulated in the tolerant group, and hub genes such as WRKY30 and KCS12 were further identified, with validation results consistent with the transcriptome sequencing trends. This study established an integrated technical approach combining precise phenotypic screening, physiological index measurement, transcriptome analysis, and gene function validation. It clarified the core physiological characteristics and molecular regulatory networks of rice salt-alkali tolerance, providing new molecular targets for the dissection of rice salt-alkali tolerance mechanisms and offering material foundations and theoretical support for molecular breeding of salt-alkali tolerant rice, germplasm development, and rice cultivation in saline-alkali lands. |