Cloning And Characterization Of Abiotic Stress Responding Genes Based On FOX-hunting System In Cotton

Cotton is one of the most important economic crops in the world.However,the current global environment is deteriorating,and soil drought and salinization are increasing.These factors have become the main environmental factors that limit cotton production.With the rapid development of biotechnology,it is of great significance to use genetic engineering technology to discover the genes related to stress resistance in cotton and obtain new cotton stress resistance varieties to increase cotton yield and cope with adversity stress.At present,the genome sequencing of Gossypium hirsutum L.,Gossypium barbadense Linn.,and Gossypium raimondii L.,etc.have been completed successively,thus cloning and characterization of cotton stress resistant genes and the exploring these functions have also become the focus of research.Our research groups constructed a homogenized cDNA library under the treatment of various stresses combining with the FOX-Hunting system and Gateway technology using the ZM12 as materials.Based on the cDNA library,we built the cotton FOX Arabidopsis overexpression library and the overexpressing strains of the cotton genes MRI2-22、AGA4-168、MRI4-470 and AGA4-119 were screened out.The genes were cloned in ZM12 and the functions of the genes were preliminarily explored.The main results are as follows:The analysis of phylogenetic tree showed that the genetic distance of MRI2-22 and MRI2-22 in Gossypium arboreum was the closest,and they both contained typical WRKYGQK domains and C2HC zinc finger structures.we found that the MRI2-22 promoter contains many cis-acting elements involved in the stress response:such as drought and ethylene etc by analyzing promoters of the genes.Overexpression of MRI2-22 in Arabidopsis was not sensitive to both NaCl and Mannitol;the germination was rapid and cotyledons became green earlier than wild type under NaCl and Mannitol treatment.qRT-PCR analysis showed that MRI2-22 was mainly expressed in cotyledons and leaf.After abiotic stress treatment,MRI2-22 was up-regulated in response to NaCl,PEG,Cold,H₂O₂,Wound stresses,and down regulated after ABA treatment.Based on the known phenotypes,a p35S::MRI2-22-GFP fusion expression vector was constructed and transformed into Nicotiana benthamiana.It was found that MRI2-22 protein was localized in the nucleus by laser confocal microscopy.Yeast experiments showed that MRI2-22 protein had transcriptional self activation activity.A pTRV2 vector of MRI2-22 gene was constructed and the silencing level was analyzed by PCR and qRT-PCR by injecting cotton cotyledons using VIGS.The MRI2-22-VIGS silencing material was found to be sensitive to NaCl after stress treatment,showing yellowing and wilting in the leaves.We also found that AGA4-168 and MRI4-470 proteins were expressed in both nucleus and cell membrane;AGA4-119 protein was mainly expressed in cell membrane by subcellular localization analysis;tissue expression analysis showed that these three genes were expressed in each tissue;AGA4-168 was expressed advantageously in cotton stems;MRI4-470 and AGA4-119 were predominantly expressed in cotyledons;stress-inducible expression analysis showed that the three genes were induced by NaCl,PEG,ABA,cold,H₂O₂ and Wound.In summary,functional verification of MRI2-22,AGA4-168,MRI4-470,and AGA4-119 in Arabidopsis thaliana revealed that they were involved in the stress response of NaCl and Mannitol and were induced by abiotic stresses in cotton.