The Roles Of Haloxylon Ammodendron HaASR2 In Plant Adapting To Drought

Climate change makes earth hotter and drier.Drought is the major threat to agriculture,human and livestock,and becomes the most important environmental factor which threatening global food security.Haloxylon ammodendron has excellent tolerance to drought,salt and barren,and widely distributes in the desert areas in northwestren China.H.ammodendron forms unique physiological and molecular mechanisms in the process of adapting to adversity,and contains abundant genetic resources for stress resistance.Therefore,it is of great significance to explore functional genes in H.ammodendron,and improve the drought resistance of crops and excellent forage grass through genetic engineering.ASR(ABA,stress and ripening induced protein)is one kind of important anti-stress protein in plants,and plays an important role in plant growth and development.Through RNA-seq analysis,we found that the expression of HaASR2 was significantly induced by drought in H.ammodendron,the results suggestted that HaASR2 was involved in the processing for adapting to drought.Under drought treatment,overexpression of HaASR2 in Arabidopsis increased relative water content,proline content,betaine content,and antioxidant capacity of leaves,in addition,the membrane damage in leaves were reduced.However,the properties and regulatory mechanisms of HaASR2 are still unclear.In this study,H.ammodendron and Arabidopsis(overexpression of HaASR2)were used as research materials.Firstly,the sequence of promoter of HaASR2 was cloned and analyzed.Next,the RNA-seq analysis was performed(WT and HaASR2-overexpression Arabidopsis plants)under simulated drought treatment.Finally,the HaASR2 protein was purificated and the secondary structure was determinated.The main research results were as follows:1.The length of HaASR2 promoter was 1332 bp,and the promoter contained drought response elements,hormone response elements and photoinduction regulatory elements.GUS staining assay showed that the promoter had driving activity,and its expression level was higher in roots,followed by leaves.By constructing expression vectors with different lengths of 5'-end deletion promoters,and transfering into tobacco by Agrobacterium-mediated method,the GUS staining assay showed that all 5'-terminated promoter with different lengths also drived the expression of GUS reporter gene.There were no dehancers and enhancers in the promoter,and the core region of HaASR2 promoter was less than 371 bp.2.The analysis of RNA-seq indicated that a total DEGs number were 1103 and406 in shoots of the control and the treatment group,respectively,and a total DEGs number were 1065 and 896 in roots of the control and the treatment group,respectively.The analysis of GO functional enrichment showed that the DEGs in the treatment group were mainly enriched in iron ion transport,reactive oxygen species response,ethylene response,abiotic stress response,etc.Further analysis showed that HaASR2 regulated drought resistance in Arabidopsis by increasing the expression of ethylene-related genes.The iron ion transport and ferritin-related genes was increasd to improve nutrient absorption and photosynthetic capacity.The POD and other antioxidant-related genes were improved to alleviate oxidative stress,and the expression of genes related to trehalose and proline synthesis were increased to enhance osmotic regulation ability.3.The prokaryotic codon optimization of the open reading frame sequence of HaASR2 gene was carried out to make the codon adaptation index CAI=1,and the optimized HaASR2 sequence was cloned into p ET28 a vector and transformed into E.coli BL21(DE3).The full length of 107 amino acids HaASR2 protein without His tag was purified by using histidine-affinity chromatography and identified by mass spectrometry.It was confirmed that HaASR2 protein was an intrinsically disordered family protein(IDPs)by using bioinformatics analysis and circular dichroism.In the purification stage,HaASR2 protein showed the metal ion binding ability,and it was speculated that it could bind metal ions to change protein conformation,playing the function of transcription factor and chaperone protein.The results showed that the promoter of HaASR2 contained a variety of cis-acting elements,which was induced by various signals,such as environment,hormones and light.Overexpression of HaASR2 in Arabidopsis regulated iron ion transport,ethylene response,osmotic regulatory substances,reactive oxygen species scavenging and other related genes.HaASR2 was an intrinsically disordered protein with metal ion binding ability,which played a protective role as chaperone protein and in the adaptation of H.ammodendron to arid habitat.