Function Analysis Of SEP4 (SEPALLATA4) Gene In Soybean

SEP4（SEPALLATA4）belongs to the MADS-box gene family.As one of the E genes of ABCDE in flower development,SEP4 plays an important role in the determination of meristem,floral organ differentiation and florescence regulation in plants.Soybean[Glycine max（L.）Merr.]is one of the most important food crops in China.It contains rich nutrients such as protein,which provides important nutritional value for humans.In order to study the biological functions of SEP4 genes in soybean,four genes homologous to At SEP4 were identified in the range of the soybean genome.The basic properties of Gm SEP4s genes were determined by phylogeny,gene structure,collinearity,secondary and tertiary structure prediction of protein,and cis-element prediction of promoter.Using q RT-PCR to analyze the floral organ expression pattern of different developmental stages of soybean W82.We also constructed overexpression and CRISPR/Cas9 gene editing vectors to transform to Arabidopsis thaliana and soybean respectively,and further explored the biological function of Gm SEP4s gene of transgenic plants with heterologous and homologous expression.The main findings are as follows:1.There are four homologous genes of At SEP4 in soybean,namely Gm SEP4a,Gm SEP4b,Gm SEP4c,all containing conserved MEF2-like MADS domain and K-domain.Gm SEP4s are closely related to At SEP4 and clustered in the same branch,while Gm SEP4a and Gm SEP4b,Gm SEP4c and Gm SEP4d are clustered in the same branch,respectively.Gm SEP4s are relatively far related to SEP-like proteins in monocots.Gm SEP4a and Gm SEP4b maybe derived from At SEP4,while Gm SEP4c and Gm SEP4d maybe derived from soybean chromosome replication.There are various light responsive elements,hormone responsive elements,and transcription factor binding sites in the promoters of SEP4s.These four genes are mainly expressed in flowers.In addition,Gm SEP4a,Gm SEP4b and Gm SEP4c are also expressed in seeds,which suggested that Gm SEP4a/b/c may be related to seed development in addition to floral organ specialization of soybean.2.The expression patterns of Gm SEP4a and Gm SEP4b,Gm SEP4c and Gm SEP4d are similar.The Gm SEP4a/b were mainly expressed in the sepals and stamens during early flowering,before-pollination,and after-pollination stages;Highly expressed in petals and stamens during the open period.Gm SEP4c/d were highly expressed in sepals and stamens at different stages,except for the stamens at the after-pollinated stage.It is speculated that these four genes may play a role in the sepals and stamens during the first three stages of flower development,and may play a role in the petals and stamens during the open period.3.Gm SEP4a,Gm SEP4b,Gm SEP4c,and Gm SEP4d proteins are all located in the nucleus.The roots,stems,leaves,pods,and inflorescences of p Gm SEP4a:GUS transgenic plants could be stained,and the promoter of SEP4a drived high expression of GUS gene in the entire Arabidopsis plant.The fruit pods and inflorescences of p Gm SEP4b:GUS transgenic plants could be stained,and the promoter of Gm SEP4b drived high expression of GUS gene in the inflorescence of Arabidopsis,while the expression in the fruit pods is lower.4.There are self activation ability of Gm SEP4b protein,while no self activation ability of Gm SEP4a/c/d protein.Gm SEP4a/c/d proteins can interact with themselves and each other.Gm SEP4 protein can interact with MADS proteins such as Gm AP1s.Gm SEP4a/b protein can interact with Gm FUL1a/b and Gm FUL3a/b proteins,and Gm SEP4b protein can also interact with Gm FUL2b protein.Gm SEP4c/d protein cannot interact with Gm FULs protein.5.Successfully constructed overexpression vectors of Gm SEP4s gene and transformed them into Arabidopsis and Soybean,respectively.Both p35S:Gm SEP4a-3Flag and p35S:Gm SEP4b-3Flag transgenic Arabidopsis plants exhibit early flowering phynotype,while p35S:Gm SEP4d-3Flag transgenic Arabidopsis plants showed no significant changes during flowering.In soybean,overexpression of SEP4a gene can observe early flowering in transgenic plants under LD and SD conditions,while overexpression of SEP4b gene in transgenic plants did not observe any changes in flowering time under LD and SD conditions.The flower organ morphology of transgenic Arabidopsis with Gm SEP4a,Gm SEP4b,and Gm SEP4d,as well as transgenic soybean with Gm SEP4a and Gm SEP4b,showed no significant changes.6.Successfully constructed Gm SEP4-1-Cas9 and Gm SEP4-2-Cas9 gene knockout vectors,the T3 and T4 targets of Gm SEP4-1-Cas9 could be effectively edited,and the T6,T7,and T8targets of Gm SEP4-2-Cas9 could be effectively edited.And sep4c,sep4d and sep4c sep4d T₂mutants were obtained through genetic transformation of soybean.Continue screening for mutants of sep4a and sep4b.Under SD conditions,sep4c,sep4d,and sep4c sep4d mutants underwent morphological changes in their calyx and petals,but their flowering time did not show significant changes.Four soybean SEP4 genes have been identified.Bioinformatics analysis and functional studies have found that Gm SEP4a genes is involved in the regulation of soybean flowering time,Gm SEP4c and Gm SEP4d genes are involved in the regulation of soybean floral organs.The role of Gm SEP4b in floral organ development remains to be studied.