Mining key drought-resistant genes of upland cotton based on RNA-Seq and WGCNA analysis

Cotton, as a globally important fiber crop, is significantly affected by drought stress during production. This study uses the drought-resistant variety Jin and the drought-sensitive variety TM-1 as test materials. Through multi-period drought stress treatments at 0d, 7d, 10d, 15d, and 25d, combined with dynamic monitoring of physiological indicators, RNA sequencing, and weighted gene co-expression network analysis, the molecular mechanism of cotton drought resistance is systematically analyzed. Dynamic monitoring of physiological indicators showed that Jin significantly accumulated proline, maintained superoxide dismutase activity, reduced malondialdehyde (MDA) accumulation, and delayed chlorophyll degradation. Transcriptome analysis revealed that Jin specifically activated 8,544 differentially expressed genes after stress, which were significantly enriched in lipid metabolism and secondary metabolic pathways. WGCNA identified co-expression modules significantly correlated with proline (r=0.81) and MDA (r=0.86) and selected the key hub gene Gh_A08G154500, which was expressed 3.2 times higher in Jin than in TM-1 at 15 days of drought stress. Functional validation suggested that WRKY22 may form a osmotic regulation-membrane protection co-regulatory network by activating proline synthesis genes (P5CS) and genes involved in the jasmonic acid signaling pathway. This study reveals, for the first time, the possible dual regulatory mechanism of WRKY22 in cotton's drought resistance, providing a theoretical basis for cotton drought-resistant breeding.