Genome-wide Identification Of TAS3 And Functional Analysis Of GhTAS3-9 Under Salt Stress In Upland Cotton (Gossypium Hirsutum L.)

Cotton(Gossypium spp.)is an important fiber and oilseed crop in the world,and salt stress seriously affects the yield and quality of cotton.The ta-si ARF produced by mi R390 induction of TAS3 gene can participate in various physiological processes in plants and play important roles in regulating plant growth and development,responding to biotic and abiotic stresses.However,no systematic identification and functional analysis of the cotton TAS3 gene family has been performed.In this study,the whole genome identification and analysis of the Gh TAS3 s in upland cotton(Gossypium hirsutum L.)was performed using bioinformatics methods,and 10 Gh TAS3 gene family were cloned and their expression patterns were analyzed.The biological functions of the Gh TAS3-9 gene under salt stress conditions were initially analyzed by Arabidopsis overexpression and upland cotton VIGS experiments.The main findings were as follows:1.Identification,analysis and cloning of Gh TAS3 gene family.Ten Gh TAS3 genes were identified in the genome of upland cotton,distributed on 10 chromosomes,and named Gh TAS3-1 to Gh TAS3-10 according to their positions on the chromosomes.A total of 65TAS3 genes were identified in 20 other species,and the phylogenetic tree was constructed together with the identified Gh TAS3 genes.The results showed that the plant TAS3 gene families could be divided into four subfamilies,and most of the TAS3 genes could be clustered together among the same species,indicating that the plant TAS3 genes are highly conserved.The public transcriptome data were downloaded for analysis and the Gh TAS3 gene expression characteristics were analyzed by q PCR experiments.The results showed that the Gh TAS3 gene family showed specificity in different tissues and organs of upland cotton,and was differentially expressed under abiotic stress conditions such as high temperature,low temperature,drought and salt stress,and the expression profiles between homologous genes had some similarity.The cis-regulatory elements of the promoter region of Gh TAS3 gene were predicted,and the results showed that the promoter regions of 10 Gh TAS3 s contained multiple stress response and other elements.These results imply that Gh TAS3 genes play an important role in regulating growth and development of upland cotton and responding to abiotic stresses.Using the genomic sequence of the upland cotton genetic standard line TM-1as a reference,we designed 10 pairs of specific cloning primers,extracted RNA from root,stem,hypocotyl,leaf and other tissue organs at the two-leaf stage of TM-1,reverse transcribed and synthesized c DNA as a template,PCR amplified the sequences of 10 Gh TAS3s,and ligated them into cloning vectors and then transformed into E.coli and sequenced them,and the sequencing results showed that the cloned sequences of 10 Gh TAS3 gene family members were completely consistent with the reference genome sequence of upland cotton genetic standard line TM-1.The cloning results further verified the reliability of the bioinformatics identification results.2.Gh TAS3-9 is positively regulating the response of upland cotton to salt stress.Based on the analysis of the expression characteristics of the Gh TAS3 gene,the Gh TAS3-9 gene was selected to further analyze its biological function under salt stress conditions.The Gh TAS3-9gene has relatively high expression levels in different tissues and organs of upland cotton,and is induced by salt stress.Its promoter region has response elements that respond to stress.Therefore,it is speculated that the Gh TAS3-9 gene is involved in the salt stress response process of upland cotton.The cloned Gh TAS3-9 gene was ligated into the p RI 201-AN overexpression vector using a homologous recombination method,and the Arabidopsis thaliana was permanently transformed by the flower dipping method using Agrobacterium mediated,and the transgenic Arabidopsis thaliana was screened and identified to obtain transgenic positive lines,and three transgenic lines were randomly selected for salt stress treatment,and the phenotype was observed and recorded and the statistical related indexes were measured.The results showed that the transgenic Arabidopsis seeds had higher germination rate,longer root length and more bioaccumulation under salt stress compared with the wild type,indicating that overexpression of Gh TAS3-9 gene could improve the salt tolerance of Arabidopsis.The p TRV2-Gh TAS3-9 gene silencing vector was constructed using a homologous recombination method,and Agrobacterium-mediated injection of upland cotton cotyledons was used to screen and identify Gh TAS3-9 gene VIGS-positive plants and perform salt stress phenotypic characterization,and the results showed that silencing the Gh TAS3-9gene severely inhibited the growth and development of upland cotton plants compared with the control,group,as evidenced by reduced plant height,reduced leaf area,and thinner stalks.And after soaking in salt solution for 4 days,the chlorosis of detached leaves became more severe.The results indicat that the silencing of Gh TAS3-9 gene made upland cotton more sensitive to salt stress.In summary,the Gh TAS3-9 gene is positively regulating salt stress tolerance in upland cotton.