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Cotton (Gossypium hirsutum L.) is a major crop and the main source of natural fiber worldwide. Because various abiotic and biotic stresses strongly influence cotton fiber yield and quality, improved stress resistance of this crop plant is urgently needed. In this study, we used Gateway technology to construct a normalized full‐length cDNA overexpressing (FOX) library from upland cotton cultivar ZM12 under various stress conditions. The library was transformed into Arabidopsis to produce a cotton‐FOX‐Arabidopsis library. Screening of this library yielded 6,830 transgenic Arabidopsis lines, of which 757 were selected for sequencing to ultimately obtain 659 cotton ESTs. GO and KEGG analyses mapped most of the cotton ESTs to plant biological process, cellular component, and molecular function categories. Next, 156 potential stress‐responsive cotton genes were identified from the cotton‐FOX‐Arabidopsis library under drought, salt, ABA, and other stress conditions. Four stress‐related genes identified from the library, designated as GhCAS, GhAPX, GhSDH, and GhPOD, were cloned from cotton complementary DNA, and their expression patterns under stress were analyzed. Phenotypic experiments indicated that overexpression of these cotton genes in Arabidopsis affected the response to abiotic stress. The method developed in this study lays a foundation for high‐throughput cloning and rapid identification of cotton functional genes. [ABSTRACT FROM AUTHOR]

• The gene GhWRKY46 , encoding WRKY transcription factor protein, was identified to be associated with drought and salt stresses. • The transcription factor GhWRKY46 locates in the nucleus and has transcriptional activation activity in yeast. • GhWRKY46 -overexpressing Arabidopsis enhanced tolerance to salt and drought as well as the expression of the stress-related genes. • GhWRKY46 silencing caused higher sensibility to salt stress and MDA level as well as lower activities of catalase and peroxidasein cotton. • GhWRKY46 positively regulates the responses to drought and salt stresses. Cotton (Upland Cotton) is one of the major fiber crops in the world. Drought and salinity, as abiotic stress factors, dramatically affect the cotton yield, However, the response processes of cotton to drought and salt stress is still not very clear. Previously, the gene GhWRKY46 , encoding WRKY transcription factor protein, was identified to possibly be associated with drought and salt pressure. In present study, the function of GhWRKY46 has been investigated in Arabidopsis and cotton. The transcription factor properties of GhWRKY46 were confirmed by its location in the nucleus and transcriptional activation activity in yeast. Overexpression of GhWRKY46 enhanced tolerance to salt and drought in Arabidopsis , conferring higher survival rates, chlorophyll contents, and biomasses in the overexpression lines under drought stress. Additionally, overexpression of GhWRKY46 enhanced the expression of the stress-related genes AtRD22 , AtCBL10 , and AtCPK3 in the transgenic Arabidopsis. Silencing of GhWRKY46 in cotton by virus-induced gene silencing resulted in higher sensibilities to salt stress and malondialdehyde contents, as well as lower activities of catalase and peroxidase compared with the control; this further confirmed previous results. Our results demonstrated that GhWRKY46 positively regulates the responses to drought and salt stresses, probably by enhancing the expression of stress-related genes. [ABSTRACT FROM AUTHOR]