Functional Analysis Of GmUFO Gene In Regulating The Floral Organ Number And Identity In Soybean

Leguminosae is the third largest family in angiosperms with important economic and ecological value.Its floral organs are rich in the morphological variation,with complex flower structure such as flower organ number variation,flower organ fusion,bilateral symmetry,and keel structure,etc.However,the evolutionary molecular mechanism of this floral organ diversity is still unclear.Soybean belongs to the butterfly flower subfamily of Leguminosae,which is an important economic crop and oil crop.It has a typical butterfly flower type,that is,symmetrical on both sides and petal-like arrangement,keel structure and wrapped two-body stamen.Soybean hybridization is an important way to solve the low yield of soybean in China,but the complex flower structure of soybean brings great difficulties to its hybrid breeding.Therefore,soybean has not yet achieved large-scale hybrid breeding.Analyzing the functions of key genes in soybean floral type provides an important theoretical basis for understanding of floral type evolution in the butterfly flower subfamily.In this study,we identified a key gene UFO(UNUSUAL FLORAL ORGANS)in soybean that controls floral organ properties and number in soybean.We clarified its spatial and temporal expression patterns at different developmental stages of floral organs,and obtained the knockout material of UFO gene by gene editing,and analyzed its molecular mechanism of regulating the attributes and number of soybean floral organs.The main results are as follows:(1)UFO gene had two homologous genes GmUFO1(Glyma.05G134000)and GmUFO2(Glyma.08G088700)in soybean,which are 78.1 % and 79.1 % similar to the UFO gene of Arabidopsis thaliana,respectively.According to the amino acid sequence alignment,it was found that both GmUFO proteins had a conserved F-box domain and three conserved motifs.The physical property analysis showed that the isoelectric point of GmUFO1 was 8.71,and the isoelectric point of GmUFO2 was 8.6.Both GmUFO proteins were hydrophobic proteins.The three-dimensional structure prediction analysis showed that the F-box structures of the two GmUFO proteins were similar,suggesting the conservative function of UFO genes in soybean.(2)The spatio-temporal expression pattern of GmUFO gene was identified by in situ hybridization.It begins to express at the initial stage of floral primordium,mainly concentrated in the central region of floral primordium.During the sepal primordium formation stage,GmUFO is transferred from the central region of the primordium to between the carpel primordium and the sepal primordium;in the subsequent petal primordium formation stage,GmUFO is mainly expressed in the petal primordium and its junction with sepal and stamen primordium.After the initiation of all floral organ primordia,GmUFO is still expressed at the boundary between petal primordia and stamen primordia,sepal primordia,and accurately regulates the initiation and subsequent development of each organ.(3)The knockout material of GmUFO gene and its trait analysis were obtained by gene editing method.Ten GmUFO1 knockout mutants,six GmUFO2 knockout mutants,and three GmUFO1 and GmUFO2 double gene knockout mutants were obtained.Among them,the number of sepals in Gmufo1 single knockout mutant was changed to 6 to 7 pieces,with a proportion of 52.82%;the number of petals changed from 3 to 7,and the number of stamens changed from 4 to 5.The development and changes of organs: vexillum folding ratio was 5.8 %,sepal petal chimera ratio was54.88 %,petal stamen chimera ratio was 5.88 %.The Gmufo2 single knockout mutant did not show significant changes in flower organ morphology compared with the wild type.In the Gmufo1ufo2 double mutation,the chimeric organ phenotype was more than that in Gmufo1,the number of sepals changed to 6,with a proportion of 28.57 %;the number of petals was changed to 3 to 7 with a proportion of 50.00%,and the number of stamens changed by 21.43 %,ranging from 4 to 5,vexillum folding ratio was 14.28 %,sepal petal chimera ratio was 84.71 %,petal-stamen chimera ratio was7.14 %,but the carpel of all mutants did not change.Through the observation of the mutant phenotype,it is speculated that the deletion of UFO gene may change the expression of genes related to control sepals,petals and stamens.Therefore,Gmufo has the characteristics of fuzzy floral organ attributes and changes in the number of internal organs.(4)The expression levels of GmAP1 a,GmAP3,GmPI＿04G and GmAG were decreased in Gmufo1 knockout plants,while the expression levels of GmAP1 a,GmAP3,GmPI＿04G and GmAG were decreased more significantly in Gmufo1ufo2 knockout plants.However,in Gmufo2 knockout plants,except for the slightly increased expression of GmAP2 and GmAG,the expression of other genes was not significantly different from that of wild type.The study of soybean UFO gene and its pathway will provide an important theoretical basis for soybean flower transformation and hybrid soybean application.