Identification And Utilization Of BR-related Genes In Soybean

As an important oil and feed crop in China,soybeans are also a high-quality source of protein,so people’s demand remains high.Soybeans have always relied on imports,and it has become very important to increase China’s soybean production.Densely planted is the main aspect of improving soybean yield,improving plant architecture is conducive to soybean dense planting,rape lactone has been found to improve plant type in other crops such as Arabidopsis thaliana and rice,while rapeolin lactone in soybean has not been reported,so this study cloned BR anabolic related genes in soybean,and explored its effect on plant architecture through overexpression and gene editing.In this study,KEGG was first used to query the anabolic pathway of brassinolide to determine the corresponding homologous genes,and the gene domain,promoter cis-element and evolutionary tree were analyzed by bioinformatics method.Secondly,the gene was cloned by homologous cloning,overexpression and gene editing vectors were constructed,mutants were obtained through soybean genetic transformation,and the plant architecture traits of the mutants were measured and analyzed,and the main results were as follows:1.Six soybean BR synthesis genes and one metabolism gene were identified by KEGG querying the rapeseedin lactone synthesis metabolic pathway.A total of 24 homologous genes were identified,with Motif 6-10 combining into a large conserved structural domain present in the genes of the BR synthesis metabolic pathway.Gm BAS1 metabolism gene lacks Motif 1-5 compared to the synthesis genes,and Gm DWF1 has a Motif structural composition that distinguishes it from other Gm DWF1 has a distinct Motif structural composition from other genes.The results of promoter cis-element analysis showed that there were a total of 55 cis-acting elements,including participation in plant hormones（gibberellin,auxin,abscisic acid,methyl jasmonate）reactions,photoresponse elements,anaerobic induction,defense and stress（hypothermia,trauma）responses,protein binding（60K,gliadin）sites,participation in meristem expression,fence tissue differentiation,MYB transcription factor binding,flavonoid synthesis,circadian rhythm regulation,etc.CYP90A1（CPD）,CYP90B1（DWF4）,CYP90C1（ROT3）/CYP90D1,CYP85A1/2（BR6ox1/2）,CYP734A（BAS1）,CYP92A6,DWF1 clustered separately,Arabidopsis,soybean and rice clustered separately.Prove that BR gene is evolutionarily conserved and functionally stable in different species.2.Gm CYP90A1（CPD）,Gm CYP90B1（DWF4）,Gm CYP90C1/D1（ROT3）,Gm CYP85A1/2,Gm CYP92A6,Gm DWF1 and Gm BAS1 genes were successfully cloned 4,3,2,4,2,2 and 3 genes,respectively,and 4,3,2,2,1,2 and 3 overexpression vectors were successfully constructed,Gm BAS1-A,Gm BAS1-B and Gm BAS1-C successfully constructed one editing vector in the 5’UTR region,and Gm BAS1-C successfully constructed one editing vector in five exons respectively.Through genetic transformation,overexpression of Gm CPD-1,3,4 obtained 18,5 and 2 positive T₁,Gm DWF4-1 obtained 11 positive T₁,Gm CYP90C1/D1-1,4 obtained 2 and 4 positive T₁,Gm CYP85A1/2-3,4 obtained 17 and 15 positive T₁,Gm CYP92A6-1 obtained 4 positive T₁,Gm DWF1-1 obtained 2 positive T₁,Gm BAS1-A,C obtained 10 and 3 positive T₁ strains,respectively.3.Overexpression of BR synthetic genes could increase plant height,and Gm CYP85A1/2-4increased significantly by 16.91%.Other synthetic genes also increased plant height to different degrees,and the number of branches increased to different degrees except Gm DWF4-1,among which Gm CYP85A1/2-4 increased the most,with an average number of branches reaching 7.6,an increase of 48%,and the number of overexpressed Gm CPD-3 plants decreased by 3.95%compared with the number of wild-type internodes,the average number of internodes was 19,and the largest increase of Gm CYP90C1/D1-2 was 19.06%,and the average number of internodes was 24.4.verexpressing Gm BAS1-C soybean plant height decreased by 70%,the number of branches was0,the petiole angle of different parts decreased by 10%-50%,and the internode length decreased by55%-82%,while overexpressed Gm BAS1-A soybean plants showed a more severe dwarf phenotype.The results showed that Gm BAS1-A played a more important role in BR metabolism than Gm BAS1-C and had a greater influence on plant architecture.There were 5 dwarfing phenotypic plants when Gm BAS1-C exon 4 was knocked out,which was contrary to BR promoting plant elongation,which was speculated to be caused by BR feedback mechanism and its effect on GA₃.High concentration of BR would in turn inhibit the expression of BR synthesis gene,reduce the synthesis of BR and GA₃,and further promote the shortening of internode,resulting in lower plant height.