Cloning,Expression Analysis And Visual Markers Development Of The Sg-5 Gene In Glycine Max

Soybean saponins are an important functional ingredient in soybeans which are mainly divided into four categories: A,DDMP,B and E.DDMP,B and E saponins have various physiological functions beneficial to the human body,while Group A saponins will lead to undesirable bitter and astringent tastes in soy products.The taste of seeds and products of soybeans accessions lacking group A saponins will be significantly improved.The GmSg-5 gene is a key enzyme gene for group A saponins synthesis.In order to improve the taste of soybean food and increase the content of saponin bioactive components,the GmSg-5gene expression pattern were analyzed.The establishment of a method that quickly identifies soybeans accessions lacking group A saponins has important significance for soybean quality breeding.Three aspects of researching were carried out in this thesis.Firstly the GmSg-5 gene was cloned from Jinyi 30,for which the gene sequence,protein sequence and promoter sequence were analyzed.Secondly,the expression patterns of GmSg-5 gene were analyzed in different tissues of root,stem and leaf,as well as different developmental stages in seeds and under the salt stress conditions by q RT-PCR technology.Through sequences alignment of GmSg-5/Gmsg5 genes which is the key enzyme gene of the group A saponins biosynthesis,the single nucleotide polymorphism(SNP)was found.Based on the SNP,specific probes for GmSg-5/Gmsg-5 genes were designed and a DNAzyme based Gap-LCR detection method for visual rapid distinction of GmSg-5/Gmsg-5 genes was developed.It has important significance for rapid screening soybeans accessions which lack group A saponins in the future.The main findings are as follows:(1)The GmSg-5 gene was cloned.Multiple hormone response elements and drought resistance response elements were found in the GmSg-5 gene promoter.The a-helix accounts for 52.25% of the secondary structure of the amino acid sequence of the GmSg-5 gene.It is speculated that the GmSg-5protein is composed of two domains,HEME and CPR.Phylogenetic analysis revealed that the GmSg-5gene was similar to Phvul005g065000,Phvul005g064100,Phvul005g063800,MTCYP72A59,and MTCYP72A219 by more than 69%,with the closest genetic relationship.(2)q RT-PCR analysis result showed that the GmSg-5 gene expression was the highest in the roots.The GmSg-5 gene expression showed a tendency of increasing at first and then decreasing at different developmental stages in seeds,and the highest at the 50 th day after flowering.The results showed that the expression of GmSg-5 gene was basically consistent with the content of group A saponins in different tissues and seeds at different development stages.(3)The expression of GmSg-5 gene in roots was suppressed in the early stage of Na Cl stress while induced in the later stage.On the other hand,Na Cl stress severely inhibited the expression of GmSg-5 gene in leaves.(4)Two sets of specific complementary probes L*,R,Ln,Rn* and L,R,Ln,Rn were designed to specifically amplify the GmSg-5 gene and the Gmsg-5 gene respectively according to the G/A variation of GmSg-5 /Gmsg-5 gene at 1127 SNP locus.(5)The visual detection method was developed under the concentration of 0.2 μmol·L-1emplate,p H8.4 LCR buffer and 1.2 μmol·L-1 probes.(6)The Gap-LCR reaction procedure was optimized,and the results showed that the Gap-LCR reaction could be completed within 1 min of extension time,which reduced the reaction time.It is found that the optimal time of detecting was 10 minutes later of the chromogenic reaction.(7)The visual specific detection method of the GmSg-5 / Gmsg-5 gene complementary probes were developed under the Gap-LCR reaction optimal system and the optimal time of detecting.In summery,the GmSg-5 gene has specificity tissue expression and spatiotemporal expression.the expression of the GmSg-5 gene is basically consistent with the content of group A saponins in different tissues and seeds at different development stages.The herein established visual DNAzyme based Gap-LCR method is capable of detecting the soybeans’ accessions of lacked group A saponins,which is rapid,simple,and economics.