Functional Analysis Of Curled-Cotyledons Mutant Of Soybean Related Regulatory Genes And Construction Of Crispr/Cas9 Mutant Library

Soybean[Glycine Max（L.）Merr.]is an important oil crop and food crop in the world,and it is one of the main sources of edible oil and protein in the world.Cotyledon is the main component of soybean seed embryo,which plays an important role in the process from seed germination to seedling.The growth state of cotyledons after germination directly affects the development status of the whole growth cycle of a plant,which will ultimately determine the traits related to resistance,yield and quality.In previous studies,we constructed a F2 generation hybridized by cco（curled-cotyledon）and Kefeng No.1 by using SSR molecular markers to assist in the selection to locate a site that controls the floding of the cotyledon,namely BARCSOYSSR₀5₀037 and Satt684 marker on chromosome 5.In this study,seven possible candidate genes were selected in this localization segment,and the expression of these seven genes in "Nanong 94-16"（WT）and cco was compared using real-time fluorescence quantitative method.Finally,based on the real-time fluorescence quantitative results and the annotation of Arabidopsis homologous genes,two genes with relatively high expression in seeds and possibly related to plant growth and development were selected,namely the △1-pyrroline-5-carboxylic acid dehydrogenase gene GmP5CDH and GmDDT1 containing the DDT（DNA binding homeobox and different transcription factors）domain.Taking GmP5CDH and GmDDT1 genes as research objects,by a series of experiments such as subcellular localization,yeast two-hybrid,and heterologous overexpression of GmP5CDH and GmDDT1 genes in Arabidopsis,to preliminary study the biological function of GmP5CDH and GmDDT1 genes,provide important information on the molecular mechanisms of curled-cotyledon mutant growth and development.Mutants are good experimental materials for plant genetics research,gene location and cloning,and gene function research,as well as important functional genomics research materials.Large-scale CRISPR/Cas9 screening with multiple mutagenesis is of great value for soybean genetic research.Therefore,this study constructed a large-scale soybean CRISPR/Cas9 mutant library of soybean seed specific expression genes.The main results were listed as followed.1.GmP5CDH belongs to △1-pyrroline-5-carboxylic acid dehydrogenase gene,containing an Aldedh domain.Tissue expression analysis found that GmP5CDH was expressed in various tissues,with higher expression in seeds 7 and 13 days after flowering.The expression level of GmP5CDH was induced by drought,salt,cold and ABA stress.GmP5CDH protein was mainly localized in the cell membrane and nucleus.Through yeast two-hybrid assay,we found that there was a fructose diphosphate aldolase Glyma.04g008300 from the leaf library and an S-adenosylmethionine synthase Glyma.17g039100 from the pod library.Heterologous expression of GmP5CDH in Arabidopsis showed that overexpression of GmP5CDH significantly reduced the germination rate of transgenic Arabidopsis,but significantly increased the total amino acid content.After transgenic Arabidopsis was treated with salt and ABA,it was found that the root length and fresh weight and the pro line content in the plant were significantly reduced.In contrast,after exogenous Pro treatment,the root length and fresh weight of the transgenic lines and the content of proline were significantly increased,and the effect on the seed germination rate was lower than WT.2.GmDDT1 is a DDT domain-containing gene in the components of the ISWI.Tissue expression analysis found that GmDDT1 gene was expressed in various soybean tissues,but was highest in seeds 7 and 13 days after flowering,and lowest in 13d pods shell.The expression level of GmDDT1 was induced by drought,salt,cold and ABA.Subcellular localization suggested that GmDDT1 is a nuclear localized protein.Through yeast two-hybrid assay,two genes from soybean leaf library were identified to interact GmDDT1,including ISWI chromatin-remodeling complex ATPase Glyma15g10370,encoding a serine/threonine protein kinase gene Glyma08g47570 and an ISWI chromatin-remodeling complex ATPase Glyma13g28720 from the pod library.Overexpression of the GmDDTl gene in Arabidopsis thaliana revealed that the transgenic Arabidopsis phenotype didn’t change significantly,but the amino acid content of the transgenic lines increased significantly.3.In this study,a total of 1193 sgRNAs were designed to target 481 genes that highly expressed in soybean seeds.Random selection of 20 monoclonals（E.coli）for Sanger sequencing found that the accuracy of sgRNA was high,reaching 95%.The obtained Agrobacterium bacterial solution can be directly transformed into soybean,which will provide valuable materials for soybean functional genome research and genetic breeding.