Identification Glutaredoxin-like Gene Family And Preliminary Functional Analysis Of Gene GhGRL26 In Gossypium Hirsutum

Objective:When a plant is under stress,it usually produces a large amount of reactive oxygen species.Excessive accumulation of reactive oxygen species can cause oxidative stress and cause serious damage to plants.Plants have evolved an effective active oxygen scavenging mechanism after a long evolution.Glutaredoxin（Gluxedoxin,Grx）is a type of small-molecule redox protein,which can regulate the redox balance of the protein to maintain the activity of the protein.In recent years,more and more glutaredoxin genes have been discovered and cloned in various plants and functionally analyzed.Glutaredoxin-like（GRL）is a newly classified type.There are few reports on the function of this new type of protein.Therefore,cloning and analysis of glutaredoxin-like gene is of great significance for further understanding the function of glutoxin family and the mechanism of plant stress resistance.Methods:According to the sequence information of the Arabidopsis thaliana glutaredoxin-like gene,the Gossypium hirsutum L.glutaredoxin-like family gene were identified and bioinformatics analyzed;VIGS technology was used to construct Gh GRL26 gene silenced plants,and the silenced plants were subjected to salt and drought stress to analyze their biological functions;At CRISPR/Cas9 gene editing technology was used to construct Cas9-Gh GRL26 editing vector and genetically transform cotton.Results:32 cotton GRL family genes were identified from the upland cotton genome database.The results show that 32 Gh GRL genes are mainly located in the nucleus,all of which have GRX-GRX-Like conserved domains.Multiple sequence alignment and conservative sequence analysis of the amino acids encoded by the Gh GRL gene found that the family members have a sequence similarity of about 31.31%,most of which contain 4 conserved motifs,and these 4 conserved motifs overlap with conserved domains;According to the phylogenetic tree of Gh GRL genes,32 Gh GRL genes can be divided into 3 subgroups.Gene structure analysis found that most of the genes in this family have no introns;chromosome mapping analysis showed that 32 genes were scattered on 19 chromosomes,and the number of Gh GRL genes on each chromosome varied significantly.analysis of expression profile data shows that Gh GRL genes are expressed in 7 tissues including roots,stems,stamens,pistils,ovary,leaves and flowers.2.Gh GRL26 gene silencing cotton plants were obtained by VIGS technology.Using q PCR technology to detect gene silencing efficiency,it was found that the expression of Gh GRL26 gene in the leaves of transgenic plants was significantly lower than that of the control,indicating that the expression of Gh GRL26 gene in upland cotton was successfully suppressed.Treat the transgenic lines with salt stress and drought stress.The silent plants were treated with salt stress,and the Gh GRL26 gene expression,chlorophyll content,CAT activity,SOD activity,H₂O₂content,MDA content and other related indicators were detected.Under salt stress,the Gh GRL26 gene expression and chlorophyll a content of the transgenic plants were significantly lower than the no-load control;The CAT activity and SOD activity of the transgenic plants were lower than the no-load control,and the SOD activity reached a significant level;The H₂O₂and MDA contents of transgenic plants were higher than those of no-load control,Among them,3.the H₂O₂content reached a significant level.Under drought stress,The relative indexes of leaf Gh GRL26 gene expression,leaf loss rate,relative water content,CAT activity,SOD activity,POD activity,H₂O₂content and MDA content were detected.The results show that,under drought stress,The Gh GRL26gene expression,chlorophyll a and relative water content of transgenic plants were significantly lower than those of no-load control;CAT activity and SOD activity were lower than that of no-load control,All reached a significant level;The contents of H₂O₂and MDA were higher than those of control plants,Among them,the H₂O₂content reached a significant level.4.Gh GRL26 gene editing embryogenic callus was obtained through CRISPR/Cas9 gene editing technology and Agrobacterium-mediated cotton genetic transformation method.Conclusion:32 Gh GRL genes were identified in upland cotton,and bioinformatics analysis revealed that these genes have similar gene motifs,gene structures,and cysteine-rich conserved domains,and these genes have different organization It is expressed in organs.The Gh GRL26 gene silenced cotton were treated with salt stress and drought stress,indicating that the Gh GRL26 gene silenced cotton plant responded to salt and drought stress.Gh GRL26 gene editing embryogenic callus was obtained through CRISPR/Cas9 gene editing technology,which laid the foundation for further exploration of Gh GRL26gene function.