Genome-wide Identification Of Aquaporin Gene Family In Cotton And Functional Characterization In Response To Osmotic Stresses

Aquaporin(AQP)is a membrane channel protein that promotes the transmembrane transport of water and other small molecules in most organisms.Studies have shown that AQPs plays an important role in response to the abiotic stress.In order to systematically explore the response mechanism of cotton AQPs under osmotic stress conditions,digging cotton resistance genes.In this study,bioinformatics analysis and expression analysis of cotton AQPs gene family were carried out using diploid and tetraploid cotton genomes and transcriptome data of upland cotton under osmotic stress conditions.By cloning the selected key genes of upland cotton,silencing vector and fluorescent expression vector were constructed,and transgenic Arabidopsis thaliana lines were obtained through genetic transformation to further verify the function of the GhAQPs gene.The main findings are as follows:1.Genome-wide indification of AQPs gene family in cotton.Through the aquaporin functional domain PF00230,a total of 215 full-length AQP genes were identified in three cotton species.Gossypium hirsutum,Gossypium arboreum and Gossypium raimondii,consist of 107,52 and 56 gene members respectively.Phylogenetic results show that cotton AQPs are divided into five subfamilies: PIP,TIP,NIP,SIP,and XIP.The protein properties,conservative site were predicted,gene structure,conserved motifs,promoter prediction,collinearity of 107 full-length AQPs of upland cotton were analyzed.The five subfamilies of cotton AQPs are divided into multiple subgroups,and the proteins in each subgroup have similar physical and chemical properties and gene structure.Most AQPs gene promoters have hormone response elements such as JA,SA,ABA and abioc stress response elements such as drought and cold and so on.2.Function characterization of AQPs in response to osmotic stress.According to the transcriptome data and real-time quantitative PCR analysis results.The expression patterns of candidate genes in response to drought and salt stresses were investigated in cotton root,steam and leaf,using q PCR analysis.,and the gene expression pattern changes as conditions change.Among them,the expressions of GhPIP1-9,GhPIP2-8,GhTIP1-1,GhTIP2-1 and other genes were significantly changed under drought stress and salt stress.Under drought and salt stress conditions,GhPIP1-9,GhPIP2-8,and GhTIP2-1 have obvious up-regulated expression in roots and stems,and downregulated expression in leaves.GhTIP1-1 is up-regulated in leaves under drought conditions,and down-regulated in leaves under salt stress conditions.3.The subcellular distribution of cotton AQPs.By cloning the AQP genes from different subgroups and constructing fluorescent expression vectors to infect tobacco transiently,observing the epidermal cells of tobacco leaves through microscope fluorescence,it was found that different AQP genes were located at different positions,among which GhPIP1-9,GhPIP2-8,GhNIP5-1 was located on the plasma membrane,GhTIP1-1,GhTIP2-1 was located on the vacuole membrane,GhSIP1-3,GhXIP1-1 was not only located on the plasma membrane,but also located in the cytoplasm.4.Functional characterization of GhAQPs by using virus induced gene silence.The VIGS technology was used to silence the endogenous GhPIP1-9,GhPIP2-8,GhTIP1-1,GhTIP2-1 of upland cotton.Compared with the control group,the silenced cotton plants showed obvious increased sensitive to drought and salt stress.Among them,the target gene-silenced cotton of GhTIP1-1 and GhTIP2-1 are the most obvious.Further experimental results showed that the roots and leaves of the silent plants had lower relative water content and higher chlorophyll content,and the difference was more obvious under drought stress and salt stress.It indicates that AQP genes are obviously involved in the regulation of osmotic stress.5.Function analysis of GhTIP2-1 in response osmotic stress.We generated transgenic Arabidopsis overexpressing GhTIP2-1 and GUS gene under the control of GhTIP2-1 promoter.Through the GUS expression,we found Under normal condition.GhTIP2-1 is highly expressed in root and leafs.Under drought and salt stress,it's down-regulated in the leaf while it kept highly express in the root.Overexpression of GhTIP2-1,confers enhanced osmotic tolerance in transgenic Arabidopsis plants.OE lines showed longer primary root length,lower hydrogen peroxide and malondialdehyde content,higher proline levels.Transcript levels of three stress-related genes were also enhanced in OE lines.This study systematically studies the distribution of cotton AQPs in the genome.Through evolutionary analysis,gene structure analysis,conservative structure prediction,promoter response element analysis,tissue expression and expression analysis under stress,the characteristics of cotton AQPs are revealed.Through functional verification of selected key genes under osmotic stress,transgenic Arabidopsis proved that overexpression of GhTIP2-1 can improve the tolerance of Arabidopsis to osmotic stress.Provide reference and reference significance.