Genome-wide Identification And Functional Verification Of Actin Depolymerization Factor (ADF) Gene Family In Gossypium Hirsutum

Actin Depolymerizing Factor(ADF),a highly conserved protein,plays an important role in the growth,development,and stress response of studied eukaryotic organisms.While there have been reports on the ADF gene family in crops such as wheat,corn,and rice,there is limited systematic research on the ADF gene family in cotton.Therefore,in this study,utilizing the transcriptomic data of upland cotton TM-1 under abiotic stress,a candidate gene Gh ADF19 associated with stress response was identified through bioinformatics analysis.The ADF gene family was further identified using whole-genome analysis,and its validation was conducted through experiments such as q RT-PCR and VIGS.The results showed that this gene significantly enhanced cotton’s resistance to a series of abiotic stresses,including cold and drought.The main research findings are as follows:From four different cotton species,namely Gossypium hirsutum L.,Gossypium barbadense Linn.,Gossypium raimondii,and Asiatic cotton,a total of 40,39,20,and 19non-redundant ADF gene sequences were identified,respectively.There were no significant differences observed in the structural domains and physicochemical properties of ADF proteins among the different cotton species.The results of the phylogenetic tree analysis revealed that the ADF family members were classified into four distinct subfamilies(I,II,III,and IV).Subfamilies III and IV comprised the majority of members,with 12 and 15 members,respectively.Based on previous studies,it is speculated that the abundance of members in these two subfamilies may be due to tandem duplication events or chromosome fragment replication events.Chromosomal mapping results showed that33 ADFs in upland cotton were located on 26 chromosomes,while 7 members were located on scaffolds,exhibiting an uneven distribution.The highest number of members was found on chromosome D09,totaling 4.Analysis of the ADF gene family in upland cotton revealed a high degree of conservation in gene structure among members within the same branch or subfamily of the phylogenetic tree.This conservation was observed in the number of exons and the lengths of introns/exons,with the majority of members containing three exons,consistent with the characteristics of the ADF family.RNA-seq analysis showed that most Gh ADFs responded to abiotic stress in different degrees,and the expression levels of Gh ADF31,Gh ADF5 and other genes were down-regulated after 1 h treatment at 4℃ and 37℃.The expression levels of Gh ADF28,Gh ADF7 and other genes increased after 1 h of low temperature treatment,while the expression levels of Gh ADF6,Gh ADF16 and Gh ADF30 increased rapidly after 1 h of treatment at 37℃,and reached the peak.At 37℃,the expression levels of most Gh ADFs members changed significantly at 1 h and 24 h,while at 6 h,12 h and 24 h under Na Cl and PEG treatments.Finally,four genes,namely Gh ADF2,Gh ADF5,Gh ADF18,and Gh ADF19,were selected through q PCR as they exhibited higher responsiveness to abiotic stress.These genes were chosen for further validation through Virus-Induced Gene Silencing(VIGS).The results of the VIGS experiments showed that cotton plants with silenced Gh ADF19 exhibited significantly poorer growth under stress conditions such as drought,low temperature,and salt compared to the control group.In summary,this study systematically analyzed the upland cotton Gh ADFs from gene structure and promoter response elements.Additionally,based on tissue expression characteristics and expression patterns,the study revealed the characteristics of the ADF gene family in upland cotton.Furthermore,the study identified the gene Gh ADF19,which exhibited a strong response to abiotic stress.These findings provide valuable insights for breeding stress-tolerant cotton varieties.