Development Of GmSg-1 Gene Visual Markers Based On HCR Reactions

Soybean saponin is one of the important secondary metabolites in soybean seeds and has many physiological functions beneficial to human body.Soybean saponins are mainly divided into A,DDMP,B and E saponins,while A soy saponins account for 68.78%and 43.17%of the total saponins in embryos and cotyledons respectively.The GmSg-1 gene is a key enzyme gene for synthesizing saponin A.By means of the anabolic pathways and qualitative and quantitative detection methods of A-type soybean saponin,it will provide the basis for the identification and breeding of type A saponins in soybean germplasm resources in the future.In this study,we determined the composition and content of soybean saponin in 68 soybean germplasm resources in Shanxi by HPLC-ESI-MS/MS method.Using this method we were able to screen specific germplasm materials with significant differences in the composition and content of soybean saponins;study the accumulation dynamics of Aa and Ab saponins and the expression of the key enzyme gene GmSg-1 during the development;use direct sequencing analysis the sequence polymorphism of GmSg-1 gene;detect of genetic polymorphisms of key enzyme gene GmSg-1 based on visualized HCR response.This method has the advantages of high sensitivity,speed and simplicity.Through this study,the main results are as follow:1.The 68 soybean materials were divided into three types:Aa type,Ab type,and AaAb type.The contents of Aa and Ab saponins in the seed embryos and cotyledons varied greatly.The variation ranges of Aa saponin content in embryo and cotyledon of Aa soybean material were 0.41-3.37 mg/g and 0.21-8.39 mg/g,respectively,and the variation ranges of Ab saponin content in embryo and cotyledon of Ab soybean material were 0.85-44.96 mg/g and 0.26-11.09 mg/g,respectively.2.The relative expression of GmSg-1 gene at different developmental stages of Jinyi 30 soybean grain increased first and then decreased,which was consistent with the change trend of Ab content during the development of Jinyi 30 soybean.3.A total of 19 SNP sites to GmSg-1 gene,wherein the amino acid variation caused by the 10 SNP site.4.Two pairs of probes GAa1,GAa2 and GAb1 and GAb2 were designed to detect the GmSg-1 gene polymorphism by HCR method.The optimal reaction system was:Tris-HCl solution pH 7.20,KC1 concentration 110 mmol/L,probe The concentration was 1.5 μmol/L,reaction time was 1 h,and the optimal observation time after coloration was 10 min.5.When the soybean material was detected by HCR method,the GmSg-1 gene fragment was 92 bp amplified by PCR,and the Aa,Ab and AaAb soybean materials were visually detected.The results were consistent with the phenotypic results and had good specificity.6.Making a gene chip to detect a class A soybean saponin by HCR reaction system.This method has the advantages of high sensitivity,rapidity and simplicity.