Cloning And Study Of Soybean GmeIF2B Gene In Drought Stress

With the global warming,extreme weather frequently appears.The global water shortage is becoming increasingly serious.Worldwide,the reduction in food production due to water shortages may exceed the total loss of output caused by other factors.China is in the process of rapid industrialization.With the intensification of industrialization,the area of available arable land is reduced,the pressure on the environment is increasing,and various extreme climates are frequently appearing,which seriously threatens our country’s food security.Drought is one of the serious environmental threats facing our country.It was found that the tolerance regulation mechanism of plants under drought stress plays an important role in improving plant drought tolerance.The use of advanced biological methods to transform crops to obtain crop varieties suitable for planting in arid and semi-arid regions with excellent agronomic traits is of great significance in actual agricultural production.Eukaryotic translation initiation factor(eIF)is a class of proteins involved in the initiation of eukaryotic translation.eIF2B is an initiation factor that converts the GDP bound on eIF2 into GTP,providing energy for the initiation of translation.Under certain external environmental stresses,the alpha subunit of eIF2 is phosphorylated,thereby inhibiting the circulation of eIF2B,thereby promoting the translation of stress-related proteins,promoting the initial expression of related molecular chaperones and amino acid synthesis and other related genes,and jointly assisting the organism Improve tolerance to adversity stress.In this study,bioinformatics analysis of the eIF2B family genes in the soybean genome,and Glyma.07G090900.2(named GmeIF2B5)was selected for in-depth analysis to explore its function under drought stress and its possible mechanism of action.The main research results of this article are as follows:1.Gene bioinformatics analysis of soybean GmeIF2B familyIn order to explore the phylogenetic relationship of the soybean GmeIF2B gene family,the bioinformatics analysis of the soybean GmeIF2B gene family was conducted,in this study.The analysis results showed that the eIF2B gene family contained 18 members in the soybean genome,and these 18 members were unevenly distributed on the 9 chromosomes of soybean.Moreover,the soybean GmeIF2B gene family could be divided into three subfamilies,and the gene structure and amino acid domains of each subfamily were different.Transient element analysis of the promoter showed that the soybean GmeIF2B gene family was a constitutively expressed gene of soybean.The most conserved GmeIF2B5 gene of the SF2 subfamily was selected under drought stress and ABA and ETH hormone treatment,which indicated the results of the gene promoter analysis.These results suggested that although the soybean GmeIF2B gene family had expanded significantly in soybeans and might be involved in many biological processes,besides translation processes.The GmeIF2B family might not respond to environmental stress through changing in nucleic acid or protein content.2.GmeIF2B5 negatively regulates soybean tolerance to drought stressIn order to indicate the function of GmeIF2B5 gene,GmeIF2B5-overexpression lines and GmeIF2B5 knockout lines were created.After drought stress,the GmeIF2B5-overexpression lines exhibited a more sensitive phenotype than the wild-type,whereas the GmeIF2B5 knockout lines exhibited enhanced tolerance to drought stress compared with that exhibited by the wild-type.In order to investigate the expression region and possible protein mechanism of GmeIF2B5,subcellular localization analysis and screening of interaction proteins for GmeIF2B5 protein were determined.The results showed that GmeIF2B5 protein was located in the nucleus,cytoplasm and cell membrane,and GmeIF2B5 protein could interact with more than 60 proteins.The GmeIF2B5 protein could not only interact with translation-related proteins,but also interact with key ETH pathway proteins GmEIL1 and peroxide by analyzing GmeIF2B5 interaction proteins.Moreover,GmeIF2B5-overexpression lines had lower peroxidase(POD)activity,while GmeIF2B5 knockout lines had higher peroxidase(POD)activity compared to wild type.These results indicated that GmeIF2B5 was an important negative regulator in the process of soybean tolerance to drought stress.It might regulate the drought stress of soybean plants in the nucleus,cytoplasm and cell membrane by assisting the transmission of ETH pathway signals and regulating the dynamic balance in ROS cells.3.Study on the function of soybean GmeIF2B5 interaction protein GmHsp90sHeat shock protein 90s(Hsp90s)was one of the most conservative and abundant molecular chaperones in the organism and an important part of the organism’s protective stress response.In order to study the function of GmeIF2B5 interaction protein GmHsp90A1,the subcellular localization analysis and screening of interaction protein for GmHsp90A1 protein were performed.The results showed that the GmHsp90A1 protein was located in the nucleus,cytoplasm and cell membrane,and the GmHsp90A1 protein could interact with its highly homologous GmHsp90A2 in the nucleus,cytoplasm and cell membrane.However,this was different from the GmHsp90A2 subcellular localization results.The GmHsp90A2 protein was located in the cytoplasm and cell membrane.These results suggested that GmHsp90A2 could be transported into the nucleus by complexing with GmHsp90A1.In this study,GmHsp90A2-overexpression lines and GmHsp90A2 knockout lines were created.After heat shock treatment,the GmHsp90A2 knockout lines exhibited a more sensitive phenotype than the wild-type,whereas the GmHsp90A2-overexpression lines exhibited enhanced tolerance to drought stress compared with that exhibited by the wild-type.Moreover,GmeIF2B5-overexpression lines had higher chlorophyll and lower malondialdehyde(MDA)content,while GmeIF2B5 knockout lines had lower chlorophyll and higher malondialdehyde(MDA)content compared with wild type.These results indicated that the interaction protein GmHsp90A1 of GmeIF2B5 could interact with GmHsp90A2 to reduce the damage of chlorophyll and maintain the stability of the membrane system,which helps to reduce the damage to plants caused by heat stress.4.GmeIF2B5 deletion can promote ABA and ethylene hormone signalingIn order to indicate the possible regulation pathways of soybean GmeIF2B,the GmeIF2B5 knockout line with better drought tolerance SP4 and wild-type was selected for transcriptome analysis under drought stress.Taking drought-treated wild-type transcripts as a reference and using | log2 FC |≥1 and P-value<0.1 as the screening criteria,a total of 1584 differentially expressed genes were obtained,including 289 up-regulated genes and 1295 down-regulated genes.GO annotation found that the differential genes involved in three major categories of biological processes,cellular components and molecular functions,among 49 subcategories.KEGG analysis found that up-regulated genes involved 18 metabolic pathways and down-regulated genes involved 22 metabolic pathways.In this study,a detailed analysis of the genes related to the ETH and ABA hormone signaling pathways in the transcriptome data revealed that the negative regulator Glyma.04G147000(named GmEBF2)in the ETH signaling pathway was significantly down-regulated in the gene-editing lines,while Regulatory factor Glyma.1 7G240100(named GmERF)was significantly up-regulated in gene editing lines.In the ABA pathway,the negative regulator Glyma.14G162100(named GmPP2C)in the ABA pathway was significantly down-regulated in the gene editing lines.The analysis results indicate that the deletion of GmeIF2B5 protein may promote the transmission of ETH and ABA signals by plants.In summary,under drought stress,GmEIL1,GmPOD and other stress-related proteins dissociate from GmeIF2B5 and perform corresponding functions,and at the same time promote the stress hormone signal transduction,so that GmeIF2B5 negatively regulates soybean tolerance to drought stress.