| 【Object】In the process of evolution,many antioxidant systems have been formed in plants to maintain and balance the redox state in plant cells.The currently known plant redox systems mainly include thioredoxin system and glutaredoxin system.Glutedoxins（GRX）are a kind of glutathione（GSH）dependent oxidoreductase commonly found in organisms,which have the activity of mercaptotransferase.In the presence of GSH,GRX can catalyze the reduction of the substrate protein disulfide bond through the single or double mercapto mechanism,and the oxidized glutathione（GSSG）generated during the reaction can complete the regeneration of GSH through the NADPH dependent glutathione reductase system.GRL members have low homology with typical GRX genes,and most of their coding proteins have eight conserved cysteine,arranged in two Cxx Cx7Cxx C clusters.At present,as a new type of GRX,there are only a few reports on the function of GRL.Cotton is one of the most important fiber crops in the world.Salt stress reduces the germination rate of cotton seeds,inhibits the elongation of main roots and the occurrence of lateral roots of seedlings,and hinders the absorption of nutrients by seedlings.Salt stress has become an important factor restricting cotton production.Therefore,exploring the function and mechanism of glutenin-like genes in cotton’s response to abiotic stress is of great significance for the cultivation of new stress-resistant cotton varieties.【Methods】（1）Based on the latest cotton genome data,the complete genome identification of the GRL genes family of two tetraploid cotton species（Gossypium hirsutum,Gossypium barbadense）and two diploid cotton species（Gossypium arboreum,Gossypium raimondii）was conducted.GhGRLs gene family was identified and analyzed based on the transcriptome data and the analysis of the results of salt stress treatment experiments.（2）Based on the changes in the expression level of GhGRL28 under salt stress,the gene was knocked out using virus induced gene silencing（VIGS）technology.The function of GhGRL28 in salt tolerance of cotton was analyzed through plant phenotype observation and enzyme activity measurement.（3）Based on the GhGRL29 dominant expressed in anthers,GhGRL29 over-expression and gene editing vector was constructed and transformed into cotton through Agrobacterium tumefaciens mediated transformation.The obtained transgenic plants and control plants were analyzed for differences in plant height,pollen viability,active oxygen species（ROS）,and carbonyl protein content under conditions of sufficient water（WW）and water deficit（WD）,respectively.The role of GhGRL29 in cotton drought tolerance and abiotic stress was further analyzed by measuring the hormone content at the growth point of the stem tip of the cotton plant,and screening the interaction protein of GhGRL29 using yeast double hybrid.【Results】（1）There were 16,16,33 and 33 GRL family members identified in G.raimondii,G.arboreum,G.hirsutum and G.barbadense,respectively.The phylogenetic tree showed that the GRL family can be divided into subgroups I-V.Collinear analysis of GRL genes of three cotton species（Gossypium hirsutum,Gossypium arboreum,Gossypium raimondii）showed that there were 14,14 and 14 collinear gene pairs between Gossypium arboreum and Gossypium hirsutum,and AD subgenomes of Gossypium hirsutum,Gossypium hirsutum and Gossypium raimondii,respectively.This indicates that the GRLs of cotton are relatively conservative during polyploidization,and GRLs undergo intensive purification selection during evolution.Although the expression level of most GhGRLs is relatively low,it has a certain degree of response to salt stress.Among them,the highly conserved GhGRL28（GH＿D10G2112）located on chromosome 10was significantly upregulated after salt stress for 1h,3h,6h and 9h,indicating that it may play an important role in cotton salt tolerance.（2）Subcellular localization showed that GhGRL28 was located in the cytoplasm.By silencing the expression of GhGRL28 gene by VIGS,compared with the control plants,the leaves of the silent plants were more wilted and the salt tolerance decreased,and the contents of SOD,POD,GSH（glutathione）,and GSH/GSSG（glutathione oxidized）in the leaves were lower than those of the control plants,while the H2O2 content,O2-content,MDA content,and GSSG content of the silent gene plants were higher than those of the control plants.q RT-PCR analysis showed that GhGRL28 silencing inhibited the expression of genes related to antioxidant enzymes and proline biosynthesis.The above results suggest that GhGRL28may regulate the salt tolerance of cotton by scavenging excess reactive oxygen species and regulating the expression of genes related to antioxidant enzymes and proline biosynthesis.（3）GhGRL29 over-expression and editing plants were obtained through genetic transformation.Under WD conditions,the anther fertility of over-expressed plants decreased significantly.The results of saffron solid green section showed that the pollen outer wall of the over-expressed plants had a small amount of spikes but collapsed and seriously vacuolized during the mature stage after drought stress.Further testing of the ROS content in the anthers of WT and over-expressed plants showed that the ROS content in the anthers of over-expressed plants was significantly lower than that of wild-type plants.It was speculated that excessive removal of ROS content led to delayed degradation of the tapetum in the anthers,leading to a decline in anther fertility.In addition,under WD conditions,the plant height of wild-type and over-expressed plants decreased by 37.9%and 18.4%,respectively,compared to WW conditions.So,the wild type control plants were more sensitive to WD stress.The results of hormone analysis of plant stem tip growth points showed that over-expression of GhGRL29can induce the synthesis of IAA,promote stem elongation and growth,while inhibiting the formation of ACC,in order to promote vegetative growth and slow down plant aging.At the same time,over-expression of GhGRL29 enhances the biosynthesis of SA and ABA,further enhancing the resistance of plants to WD stress.The deletion of GhGRL29 leaded to extremely weak growth potential,which makes it difficult for plants to grow tall and exhibits complete abortion.This also indicates that GhGRL29 plays an important regulatory role in the growth and development of cotton plants.In order to further explore the mechanism of GhGRL29participating in cotton response to drought stress,GhGRL29 protein was constructed into yeast vector p GBKT7 to screen a yeast library.Thirty-two proteins that may interact with GhGRL29 were screened,and four interacting proteins were preliminarily analyzed:MIOX1（Gh＿A05G1099）,GhWRKY33（Gh＿A03G0210）,FBA（Gh＿A04G0544）,ZAT12（Gh＿D13G2392）.The results lay a foundation for further studying the molecular regulatory mechanism of GhGRL29.【Conclusion】33 GhGRLs were identified in upland cotton.Among them,GhGRL28 can regulate cotton salt tolerance by clearing excess reactive oxygen species in the plant and regulating the expression of antioxidant enzymes and proline biosynthesis-related genes.Over-expression of the GhGRL29 gene leads to excessive clearance of ROS content,resulting in delayed degradation of the anther tapetum and a decline in anther fertility.GhGRL29 may enhance cotton drought resistance by increasing hormone content.The phenotype of GhGRL29 knockout plants indicates that the decreased expression level of GhGRL29significantly affects plant growth and development.Therefore,both high GhGRL29 expression levels（increased resistance but decreased fertility）and low expression levels are not adverse for cotton plant growth.Maintaining a balance of plant-related gene expression levels and appropriately increasing the expression level of GhGRLs are of great significance for cotton stress tolerance.This study provides a theoretical basis for further studying the functions of the GRL family genes as well as the molecular mechanisms underlying the growth and development of cotton under abiotic stresses such as drought and the regulation of cotton stress tolerance. |
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