| At present,soybean [Glycine Max(L.)Merr.] is an important oil,edible and feed crop in the world,and is the main source of protein and oil for mankind.Wild soybeans [Glycine soja Sieb.& Zucc.] have outstanding genetic diversity,which has great application value for modern soybean breeding.It can also provide rich research materials for the cultivation of new soybean varieties,and contribute to the development of the soybean industry.Provide strong support for innovation and development.Yield breeding is the focus of soybean breeding and the focus of soybean production development.Improving yield has always been the primary goal of genetic breeding.Grain weight is one of the important agronomic traits that determine soybean yield,and generally shows a positive correlation with yield.The grain weight is affected by many factors.The study of grain weight from the genetic level provides an effective research idea for soybean breeding.It has certain practical significance to increase the soybean yield on the basis of increasing the grain weight.Heilongjiang has abundant wild soybean resources,which can provide abundant materials for soybean genetic improvement and improved seed breeding.The predecessors used the natural population composed of 249 wild soybean resources in Heilongjiang Province as experimental materials,investigated the 100-seed weight traits of the three years in 2012,2013,and 2018,and carried out genome-wide association analysis research on the basis of collating relevant data.Screen the SNP sites that have been repeatedly located for many years,look for candidate genes 50 kb before and after the candidate SNP site,perform QRT-PCR analysis on the candidate genes and refer to function annotations to finally determine the two candidate genes GsORF328 and GsORF62 for subsequent molecular function studies,Through bioinformatics analysis,tissue expression,differential expression of extreme materials,subcellular localization,transformation of Arabidopsis thaliana and other ways to study the functions of these two candidate genes,preliminary study of their regulation mechanism on yield,so as to improve soybean Yield and exploring wild soybeans provide theoretical basis for the evolution of cultivated soybeans.The main findings are as follows:1.GsORF328 is a gene encoding MLP(Major latex protein)protein in wild soybeans.It belongs to the MLP subfamily of the Bet＿v＿1 protein family and contains a Bet＿v＿1domain.Linkage disequilibrium analysis showed that there are a total of 10 SNP markers in the gene,of which 9 SNP markers are linked.Haplotype analysis showed that the gene had4 haplotypes,and H1 was the best haplotype.Tissue expression analysis found that GsORF328 was expressed in all tissues,and the pod was the most prominent expression site.Based on the results of the expression pattern of pod tissues,the gene was expressed in DAF 7-day pods in materials with heavier 100-seed weight,and in the middle and late stages of pod development,DAF15,DAF25,DAF35,and DAF45-day pods had higher100-seed weight.The expression is higher in small extreme materials.The extreme material HAAS＿081 and HAAS＿187,which have a smaller 100-seed weight at 35 days of DAF,had higher expression levels.According to the results of subcellular localization analysis,this protein is a nuclear-plasma membrane colocalization protein.According to the results of yeast transcription activation activity detection and analysis,this type of protein does not show the characteristics of transcription activation activity,and the toxicity detection result of the protein is non-toxic.The GsORF328 gene is heterologously expressed in Arabidopsis.Compared with the wild type,overexpression of GsORF328 Arabidopsis changed a series of phenotypes,including leaf type,plant height,number of branches,stem thickness,number of siliques,thousand-grain weight,and Seed weight per plant.2.As one of the representative genes of the MADS family,GsORF62 will show the common characteristics of the MADS domain.From the results of tissue expression pattern analysis,it can be seen that leaves are the most prominent part of the gene expression.Relatively speaking,the expression level of seeds,pods and other parts is low.Based on the analysis results of the pod tissue expression pattern,the gene expressed in DAF7 and DAF15-day pods in materials with heavier 100-seed weight.The extreme material changes of DAF25 were not obvious in the middle stage of pod development.The expression level is higher in extreme materials with smaller 100-grain weight.In DAF 35-day seeds,the expression level was higher in extreme materials with heavier 100-seed weight.GsORF62 is a transcription factor,and the nucleus is the location of its protein.Transcription activation activity detection The full length of the protein does not have transcription activation activity,and the toxicity detection result of the protein is non-toxic,which can be used for subsequent screening experiments.The phenotype of transgenic Arabidopsis overexpressing GsORF62 was identified.Compared with the wild type,the transgenic plants changed in leaf type,silique number,thousand-seed weight,and seed weight per plant. |
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