Functional Characterization And Study On Mechanism Of Soybean GmFBL144 Gene

At present,soybean is an indispensable raw material for high protein food and healthy vegetable oil.As the largest soybean consumer,more than 80%of soybeans come from imports,which greatly affects China’s food security.Therefore,it is extremely important to increase domestic soybean production and reduce excessive dependency on imported soybeans.However,soybean yield is affected by many factors,among which drought stress is one of the main factors for soybean yield reduction.Based on laboratory transcriptome data of soybean under drought stress,it was found that an F-box gene,GmFBL144,responded to drought stress.In order to study the function of soybean GmFBL144 gene,the bioinformatics analysis of soybean GmFBL144 gene was carried out in this study.And its expression pattern,promoter activity and subcellular localization were analyzed.Furthermore,the abiotic stress analysis and development phenotype identification of transgenic Arabidopsis and soybean were carried out to study the function of soybean GmFBL144 gene.In addition,in order to further study the mechanism of GmFBL144 gene,we constructed the c DNA library of soybean in the yeast nuclear system,and identified the interaction protein GmsHSP of soybean GmFBL144 by yeast two-hybrid and Bi FC assay.On this basis,GmsHSP gene expression pattern analysis,subcellular localization and functional identification were carried out.And the action processes and stability of GmFBL144were studied.The major results in this study are as follows:1.Bioinformatics analysis of soybean GmFBL144 gene showed that GmFBL144protein was an unstable hydrophilic protein.There were four drought responsive elements,one stress responsive element,one gibberellin responsive element and two cis-acting elements involved in Me JA response in the promoter region of GmFBL144gene.2.The expression analysis of GmFBL144 gene showed that under drought stress,the relative expression of GmFBL144 gene first increased and then decreased,and it was inhibited（down-regulated）after 1 h.Under salt and alkali stress,the relative expression level of GmFBL144 also showed a trend of first increase and then decrease.After 6 h treatment with different hormones,the relative expression of GmFBL144 gene was significantly down-regulated（except ZR treatment）.In addition,promoter activity analysis showed that GmFBL144 gene promoter was active in various tissues（roots,stems,leaves,flowers and pods）of plants.3.Under drought and salt stress conditions,the content of reactive oxygen species（ROS）and malondialdehyde（MDA）in GmFBL144-overexpression Arabidopsis thaliana was higher than that in WT and empty control,resulting in more serious damage to leaves than WT and empty control,and reducing the tolerance of plants to drought and salt stress.Under normal conditions,the bolting,flowering and maturation time of GmFBL144-overexpression Arabidopsis were delayed 3-4 days later than WT and empty vector,and the plant height of transgenic Arabidopsis was higher than WT and empty vector.4.Under drought stress conditions,the content of ROS and MDA in GmFBL144-overexpression soybean was higher than that in WT,resulting in more serious damage to leaves than WT,and reducing the tolerance of soybean to drought stress.Under normal conditions,the plant height of GmFBL144-overexpression and dominant inactivation mutantΔF was shorter than that of WT control.In the field experiment,GmFBL144-overexpression andΔF delayed the growth period of soybean and increased grain size.5.We found that GmFBL144 protein was mainly localized in the nucleus by subcellular localization assay.Thus,we constructed a soybean c DNA library of yeast nuclear system and screened the interaction protein GmsHSP by yeast two-hybrid system.The interaction between GmFBL144 and GmsHSP was verified by yeast two-hybrid and Bi FC assay.6.The expression analysis of GmsHSP gene showed that the relative expression level of GmsHSP was high in leaves and pods,and it was induced by drought and salt stress,and responded to multiple hormone treatments,indicating that GmsHSP may be involved in regulating plant growth and development and stress response.In addition,the functional study of GmsHSP gene found that GmsHSP gene was a positive regulator of drought and salt stress.7.GmFBL144 combined with Skp1,Cullin1 and Rbx1 through the F-box domain to form SCF^FBL144（E3 ubiquitin ligase）,ubiquitinized the target protein GmsHSP,and then it was degraded through 26S proteasome,resulting in plant sensitivity to drought and salt stress.