Transformation Of GmSUMO2 In Soybean And Functional Analysis Involved In ABA And Drought Response

SUMO is a small protein that is structurally similar to ubiquitin.It mainly participates in the protein post-translational modification process by covalent binding to target proteins.SUMO modification is a reversible cascade reaction,the SUMO protein is hydrolyzed by SUMO protease to expose the diglycine structure and form a functional SUMO protein;SUMOyaltion will change the localization,stability and activity of these modified substrates and participating in the regulation of multiple biological processes.In Arabidopsis,SUMO play important roles in plant growth and developmental regulation,environmental stress response and hormone signal transduction.In previous studies,it was known that SUMOylation occurred rapidly in soybean under high temperature,high salt and ABA stress treatments.But,the function of SUMO under abiotic stress in soybean has not been reported.Therefore,this study will improve the knowledge of plant abiotic stress signal transduction pathway from the perspective of protein post-translation modification through the preliminary study of GmSUMO2 gene function,and provide plant materials for soybean molecular breeding,which has theoretical research significance and breeding application value.In this research,GmSUMO2 gene was cloned and analyzed.In order to facilitate the follow-up study of identification of the SUMOylated substrates in soybean,to 94 th amino acid histidine(H)of GmSUMO2 was mutated into arginine(R),which would add a R loci of trypsin digestion to facilitate mass spectrometry analysis.Meantime,it will distinguish ubiquitin and SUMOylated substrates.After cloning of GmSUMO2 and GmSUMO2(H94R),p TF101-6×His-GmSUMO2 and p TF101-6×His-GmSUMO2(H94R)were constructed.Overexpression soybean hairy roots were obtained by Agrobacterium rhizogenes mediated method.The functions of GmSUMO2 gene in soybean under ABA and mannitol stress treatment were analyzed.In addition,GmSUMO2 gene was transformed into soybean cotyledonary nodes using Agrobacterium tumefaciens mediated method.PCR positive soybean plants were obtained,which provided plant materials for further study.The main results obtained in this study were as follows:(1)GmSUMO2 gene was cloned.Through the homology comparison of soybean GmSUMOs gene sequence,the GmSUMO2 and At SUMO1 were highly similar.By analyzing the existing RNAseq data in the database,it was found that GmSUMO2 gene was expressed in different tissues of soybean.Promers of GmSUMOs contained several abiotic stress response elements by cis-element analysis.The expression patterns of GmSUMO2 was analyzed by q RT-PCR,which confirmed the notion that GmSUMO2 was responsive to salt,heat,ABA and drought at the transcriptional level.(2)The 6×His tag sequence was fused at the 5 terminal of GmSUMO2 gene,and the diglycine structure of GmSUMO2 protein in the C terminal was exposed.At the same time,specific primer PCR amplification was used to obtain the point mutation form GmSUMO2(H94R).p TF101-6×HisGmSUMO2 and p TF101-6×His-GmSUMO2(H94R)plant expression vectors were constructed and transformed into Agrobacterium rhizogenes and Agrobacterium tumefaciens.Agrobacterium rhizogenes K599 was used to induce the hairy root of soybean,and overexpression of GmSUMO2 gene in transgenic hairy roots was confirmedby PCR and Western blot.(3)The transgenic soybean hairy roots were treated by 0 ??,15 ??,25 ?? ABA and 100 m M mannitol.Phenotypic analysis showed that soybean hairy roots overexpression of GmSUMO2 and GmSUMO2(H94R)were sensitive to ABA treatment.The results of q RT-PCR confirmed that GmSUMO2 expression was significantly up-regulated in the transgenic hairy roots,and the expression level of GmSUMO2 in transgenic hairy roots was greater than control hairy roots under ABA treatment.Meanwhile,q RT-PCR analysis was performed to detect the expression levels of ABI3/5,Sn RK1.1/1.2 which involved in ABA transduction pathways.The results show that the expression level changes of ABI3/5 were similar,which showed enhanced expression under ABA treatment in control soybean hairy roots.But the expression levels of ABI3/5 were not significantly changed under the treatment of 15 ?? ABA in transgenic soybean hairy roots,which were much lower than in control hairy roots.When the ABA concentration was 25 ??,the expression leves of ABI3/5 were up-regulated significantly.Besides,the expression levels of Sn RK1.1/1.2 were not changed before and after ABA treatment in control hairy roots,which showed enhanced expression levels with the increase of ABA concentrations in transgenic hairy roots.The gowth of transgenic hairy roots were inhibited under mannitol treatment compared with control hairy roots.q RT-PCR analysis indicated that the expression level of DREB2 A was enhanced significantly under mannitol treatment in transgenic hairy roots,and the other maker gene b ZIP1 showed no significant difference under mannitol treatment in both control and transgenic hairy roots.Moreover,there was no significant differences between GmSUMO2 and GmSUMO2(H94R)overexpression soybean hairy roots either on phynotypes or on the expression levels of marker genes under stress treatment.It indicated that the point mutation has no effect on the function of GmSUMO2.(4)Genetically modified plants of soybean variety "DN50" were obtained by Agrobacterium tumefaciens mediated transformation method.Two positive soybean plants transformed with p TF101-6×His-GmSUMO2 and two positive soybean plants transformed with p TF101-6×HisGmSUMO2(H94R)were obtained.At present,14 seeds of T1-generation of transgenic soybean have been harvested,which provided preliminary materials for further studies.