Localization And Preliminary Functional Validation Of GHiGT-51:A Key Candidate Gene In Cotton’s Response To Drought Stress

[Objective]Xinjiang,a key cotton-producing region in China,grapples with frequent drought disasters due to its meager rainfall and high evaporation rates.Beyond devastating the agricultural ecosystem and shrinking crop cultivation areas,drought exerts a severe toll on cotton growth,development,and yield formation.Consequently,it stands as a formidable obstacle hindering cotton production and sustainable development.The intricate regulatory network governing cotton response to drought stress remains elusive,demanding exploration.Hence,uncovering novel drought tolerance genes in cotton and unraveling the underlying mechanisms that dictate its response to drought stress assume paramount importance.This pursuit not only facilitates the breeding of resilient cotton varieties but also fosters the sustainable advancement of cotton production.[Methods]In this study,we selected two cotton cultivars with contrasting drought tolerance,namely Xinluzao 13（Z13）and Xinluzao 22（Z22）,and subjected them to drought stress during the seedling stage using both hydroponic and soil-based methods.We measured morphological,physiological,biochemical,and photosynthetic parameters to assess drought tolerance in cotton and identify key indicators that respond to drought stress.Furthermore,we constructed an F₂ mapping population consisting of 301individual plants and employed bulk segregant analysis and linkage analysis to identify candidate genomic regions and genes associated with drought resistance in cotton.Additionally,we performed virus-induced gene silencing on GhiGT-51 and conducted whole-genome identification and bioinformatics analysis of the Trihelix transcription factor family（Trihelix family）to gain insights into their role in drought tolerance.[Results]（1）Under drought stress,Z13 exhibits superior drought tolerance compared to Z22.Z22 demonstrates higher stomatal density（SD）,resulting in accelerated rate of water loss（RWL）and wilting,alongside reduced water use efficiency（WUE）.Physiologically,Z22 shows increased levels of malondialdehyde（MDA）and decreased antioxidant enzyme activity compared to Z13.（2）Using BSA-seq and linkage analysis,we identified a drought-related QTL:q DLW-A12-1（186.32 kb）in cotton.Within this genomic interval,we conducted drought-induced expression analysis on eight candidate genes.Among them,GhiGT-51 showed induced expression under drought stress,with significantly higher relative expression levels in Z13 compared to Z22.These findings suggest the potential involvement of GhiGT-51 in regulating cotton response to drought stress.（3）Functional analysis using VIGS uncovered the negative regulatory role of GhiGT-51 in SD.Silencing the GhiGT-51 gene led to a significant increase of SD in plant,degree of leaf wilting（DLW）and RLW,and at the same time reduced the activity of antioxidant enzymes under drought stress in cotton,reducing the ability to remove reactive oxygen species and osmoregulation in the body,making cotton Drought tolerance is reduced.（4）To further understand the evolutionary selection pressures on the GhiGT-51 gene,we identified 100,100,51,and 49 members of the Trihelix family from Gossypium hirsutum,Gossypium barbadense,Gossypium arboreum,and Gossypium raimondii,respectively.The Trihelix family was categorized into subfamilies including SIP1,SH4,GTγ,O,GT-1,and GT-2.Evolutionary pressure analysis revealed that Trihelix genes underwent purification selection during the evolutionary process.Analysis of promoter cis-acting elements,tissue-specific expression,and expression patterns under stress conditions in upland cotton Trihelix family members indicated that GhiGT-13 and GhiGT-50 might play crucial roles in cotton’s response to drought stress.[Conclusion]The GhiGT-51 gene negatively regulates stomatal density in cotton,and suppressing its expression significantly reduces cotton’s drought tolerance.Through analysis of the Trihelix gene family,we identified GhiGT-13 and GhiGT-50 as potential candidate genes involved in cotton’s response to drought stress.These findings provide candidate genes for the development of drought-resistant cotton varieties and hold significant theoretical and practical implications for the exploration of drought-related genes and molecular mechanisms in future studies.