Genome-wide Identification,Cloning And Functional Analysis Of Salt Stress-related Wrky Transcription Factors In Gossypium Aridum

To survive and adapt in the adverse environment,such as drought and high salinity,plants have evolved a series of complex and effective regulatory mechanisms that acting at the cellular,molecular,physiological and biochemical levels.The existing study indicated that this regulatory mechanisms from self-evolution operated by transcription activation or inhibition of corresponding genes.In these process,transcription factors play important roles.They activate or inhibit the expression of target genes by interacting with other related proteins or themselves.Recent studies suggested that a lot of WRKY family members involved in the response of plant to abiotic stresses.Among IFs,the WRKY description factors have become an important topic in plant resistance function studying in past decades.Cotton genome is large and complex.Moreover,the transcription factors are numerous.Therefore,the current research is limited to bioinformatics analysis and of the functional prediction and so on.And it is not clear that the structure of a single gene,physiological and biochemical functions,regulatory networks under salt stress in cotton.In this study,the diploid wild-species Gossypium aridum was used as experimental materials for identification,cloning and functional analysis of salt stress-related WRKY transcription factor in Garidum in view of it's remarkably tolerant to salt stress.1.The total of 109 WRKY genes were identified from the Garidum transcription databases(root and leaf)by Bioinformatics analysis.According to the 126 WRKY domain of 109 GarWRKY genes and the WRKY domain features of GarWRKYs,the phylogenetic tree was constructed and 109 GarWRKY genes were classified into three groups,designated Group?to Group?.17 GarWRKYs were assigned to Group?.80 GarWRKYs were assigned to Group?.12 GarWRKYs were assigned to Group?.GarWRKY genes are distributed on all 13 Garidum chromosomes and the majority are concentrated in the end of chromosomes.2.28 salt stress-related GarWRKY genes were identified and cloned based on the digital gene expression profile analysis of G.aridum during different stages of salt stress in roots or leaves.GenBank accession number are KM438453-KM438480.The ORF length ranged from 474 bp to 1848 bp with an average length of 1048 bp.The identified GarWRKY genes encode proteins ranging from 157 to 615 amino acids(aa)in length with an average of 348 aa.All 28 salt-responsive GarWRKY proteins were predicted to be located in the nucleus.Using RT-PCR,we examined the expression patterns of GarWRKY genes in plants grown under normal growth conditions in seven different tissues including root,stem,leaf,petal,stigma,stamen and calyx tissue.The results showed that not all WRKY genes are expressed in all detected tissue.27 GarWRKY genes were expressed in roots,while 6 genes were expressed in all tissues.3.qRT-PCR was used to confirm GarWRKY levels of expression in roots and leaves after salt stress treatment.Most GarWRKY genes in roots were activated within 1 h following salt treatment with peak expression during the middle period(12 and 24 hours)a nd a return to basal levels during the latest period(72 h).However,in leaves,the majority of the analyzed WRKY genes were activated later than 6 h after salt treatment,with peak expression during the latest period(72 h).The analysis of WRKY gene expression under salt stress indicating that plants perceive salt stress and activate their defense machinery rapidly in roots.4.Three genes,GarWRKY5 and GarWRKY17(up-regulation after salt stress)and GarWRKY22(down regulation after salt stress)were selected to generate overexpression transgenic Arabidopsis plants to further evaluate their function in response to salt stress.They were cloned into the plant expression vector pCAMBIA2301.and transgenic Arabidopsis plants lines were obtained by floral dip method mediated by Agrobacterium tumefaciens-mediated transformation.The phenotypic,physiological and biochemical analysis of T3 generation transgenic Arabidopsis lines indicated that the over-expression of genes GarWRKY5 and GarWRKY17 can remarkably improvewill improve the tolerance of transgenic Arabidopsis under salt stress,but the overexpression of GarWRKY22 can increase the sensitivity to salt stress in transgenic Arabidopsis.The VIGS phenotype analysis showed that the mutant cotton plants plants of GarWRKY5 and GarWRKY17 genes displayed more serious salt harm under salt stress than the control group,while no significant difference was observed between control and the GarWRKY22 mutants.5.The transcriptome of GarWRKY17-overexpressing transgenic Arabidopsis lines under normal and salt stress conditions were sequenced.A total of 11 downstream target genes salt stress-related of GarWRKY17 were screened in G.aridum.RT-PCR was performed to analyze the expression pattern of these genes under salt stress.Finally,several regulation networks were predicted as follows:(1)The expression of NRT1.8(NO3-transporter)was been activated.On the one hand,the NO3-was transported into the vacuole to keep the ionic equilibrium to improve the salt tolerance of plant;on the other hand,NRT1.8 can promote the nitrogen absorption of plant and synthesize a number of organic substances for osmotic adjustment by the amino acid metabolism pathway and the secondary metabolic synthesis pathway,which increased the salt tolerance of plants;(2)Ca2-transporting ATPase was activated.The Na+/K+ reverse ion transporter pumped out excess Na+ to maintain the ionic balance in cells,protecting plants from the damage though The SOS pathway;(3)Under normal conditions,MYB102,NAC3 and GarWRKY17 interacted with each other and inhibited the expression of GarWRKY17.When plants received salt stress signal,the chimeric repressor of MYB102 and NAC3 was activated and accumulated.Finally the interaction of MYB102,NAC3 with GarWRKY17 was disrupted.Thus,the GarWRKY17 can express and activate the downstream DREB-and ZAT-related genes to improve the salt tolerance in plant;(4)In addition,salt stress signal enhance the interaction of GarWRKY17 with GarWRKY28.This combination regulation enhanced the ability of responding to salt stress to improve the salt tolerance of plants.6.The DGE analysis and RT-PCR was performed in GarWRKY22-overexpressing transgenic Arabidopsis lines under normal and salt stress conditions.14 downstream salt stress-related target genes were obtained in Garidum.We predicted possible regulation pathways to regulate the salt tolerance of plants as follows:(1)A number of organic substances were synthesized for osmotic adjustment by the amino acid metabolism pathway and the secondary metabolic synthesis pathway;(2)The activity of related electron transporter and enzymes are regulated to participated in the response to salt stress by the photosynthetic pathway;(3)Some genes encoded hormone,such as ethylene,were inhibited to increase the sensitivity to salt stress in plant by hormone signal transduction pathways;(4)Interacting with other some transcription factors,such as Zinc-finger proteins and LBD proteins to further regulate the salt stress-related target genes.(5)Moreover,Downstream salt stress-related genes was activated through MRPK activity.