Cloning And Functional Analysis Of Drought Stress Response Gene TaSADR1 In Wheat(Triticum Aestivum L.)

Drought stress is the main factors that reduce growth,yields and quality in wheat.several RING finger proteins function as E3 Ub ligases in response to development and abiotic stress in plants.Os SADR1 was previously identified as a drought,salt and ABA stress-induced RING finger protein gene,and negatively regulates drought response in rice.Herein,we cloned and identified the orthologue gene of Os SADR1 in wheat(Triticum aestivum L.)and called Ta SADR1.Then we analyzed the gene structure,promoter sequences,amino acid sequence and evolutionary relationships of the Ta SADR1.Besides,the q RT-PCR assay,prokaryotic expression assay,self-ubiquitination assay,subcellular localization assay and yeast self-activation assay was continued to analyze the gene expression and protein function.At last,the transgenic Arabidopsis was created and was used to analyze the function of Ta SADR1.Now the results as follows.1.the Ta SADR1 gene was cloned and identified,containing a 1407-bp-long open reading frame(ORF)with a predicted protein of 486 amino acids,at the N-terminal of the protein contains a C3HC4 type RING finger conserved domain;and the gene was composed of 14 exons and 13 introns.The evolutionary relationships analysis shown Ta SADR1 protein is in the same branch with the E3 ubiquitin-protein ligase from Aegilops tauschii,Triticum Urartu,Hordeum vulgare,Zea mays,Setaria italica,Setaria viridis,Oryza sativa and has high homology;Among of those proteins,Ta SADR1 had closest relationship with the protein from Aegilops tauschii.Quantitative analysis demonstrates the expression level of Ta SADR1 gene increased with time at seedling stage.Besides,Tissue-Specific expression analysis indicated the expression levels of Ta SADR1 were relatively higher in the steam compared with the root.however,the expression levels were lower in the leaf and panicle.Abiotic stress indicated Ta SADR1 was highly induced by ABA and PEG6000,then was salt,cold and the last was heat.2.The prokaryotic expression analysis of Ta SADR1 reveals that the product of recombinant plasmid had a molecular mass of 105 k Da,and the product of p Cold–TF vector had a molecular mass of 55 k Da,the molecular mass of proteins was consistent with the theoretical molecular mass.The self-ubiquitination assay shown Ta SADR1 protein has self-ubiquitination activity when E1,E2 enzymes and Ub existed.The subcellular location of Ta SADR1 protein shown that the fluorescence signal of Ta SADR1-EGFP fusion protein was present in nucleus and the 35S::EGFP mainly distributed at the plasma membrane.The yeast self-Activation analysis of Ta SADR1 shown the full-length coding sequence of the Ta SADR1 gene without self-Activation activity.3.Germination experiments of transgenic Arabidopsis thaliana showed that the growth,root length and germination rate of transgenic Arabidopsis thaliana were significantly lower than that of wild-type Arabidopsis thaliana under drought(simulated by mannitol).Germination of transgenic Arabidopsis thaliana was significantly inhibited under the existence of ABA.These results indicate that overexpression of Ta SADR1 gene reduces Arabidopsis resistance to drought and salt stress,and increases its sensitivity to ABA.4.Under drought stress,the leaves of transgenic Arabidopsis thaliana plants were wilted and stalks were dried up after ten days of drought,while the wild-type Arabidopsis thaliana grew better than transgenic Arabidopsis thaliana.Besides,the wild-type Arabidopsis thaliana recovered and transgenic Arabidopsis thaliana withered after rehydration.Physiological index analysis showed that the overexpression of Ta SADR1 gene decreased the water retention ability of Arabidopsis thaliana after drought treatment,and the permeability of cell membrane was also increased,making it easier for cell electrolytes to leak;Plants are more vulnerable when their defenses against drought stress.The production rate of osmotic regulatory substances decreased and the homeostasis of cells were broken.The activity of peroxidase decreased,and the harmful substances in the cell could not be removed in time.Quantitative analysis of four abiotic stress response genes(RD29A,RD29 B,DREB2A and P5CS1)of wild-type and transgenic Arabidopsis thaliana in normal culture and drought treatment indicated that overexpression of Ta SADR1 gene inhibited the response of the four abiotic stress response genes to drought stress.In a word,Ta SADR1 gene played a negative regulatory role in drought stress in Arabidopsis.In conclusion,we successfully cloned the Os SADR1 homologous gene Ta SADR1 in wheat.The gene was localized in the nucleus and response to drought and ABA treatment.The protein of Ta SADR1 gene had E3 ubiquitin ligase activity,which function as a negative regulator of drought stress.