Study On The Mechanism Of Synthesis And Metabolism Of Soyasaponins

Glycine max is one of the most important crops in China.Soyasaponins are important secondary metabolite in soybean seeds.With the deepening of research,it has been found that there are many beneficial functions to the human body,so people pay more attention to it.However,there are few studies on the biosynthesis mechanism of soyasaponins.In this study,72 soybean varieties were used as the research object,the content of soyasapogenol in 72 soybean varieties was determined by high performance liquid chromatography(HPLC),and the relationship between the content of soyasapogenol and agronomic traits was analyzed.The different of grain weight,seed coat color,the ecological types and content of soyasaponins in soybean varieties were evaluated.Soybean varieties with different content of soyasaponins were selected,and the content of soyasaponins in the grains were determined by high performance liquid-electrospray ionization tandem mass spectrometry technology(HPLC/ESI-MS/MS),and transcriptome sequencing,screening differentially expressed genes involved in metabolism of soyasaponins.The expression pattern of differentially expressed genes in soybean grain development and different tissues was analyzed by qRT-PCR.In this study,we explored the mechanism of synthesis and metabolism of soyasponins from the transcriptome and metabolism level,in order to explore the key enzyme genes of soyasaponin synthesis,and laid the foundation for the genetic improvement of soybean.The results are as follows:1.The content of soyasapogenol in 72 soybean varieties was determined by HPLC,whose results showed the variation range of soyasapogenol A,B and total soyasapogenol is 0.19-1.54 mg·g-1,0.63-2.69 mg·g-1 and 0.92-4.02 mg·g-1,respectively.There are differences between the content of soyasapogenol in different soybean varieties.The total soyasapogenol content in soybean of small,black seed coat,wild type is the highest.Inversely,the large,yellow seed coat,cultivatied soybean varieties is the lowest.In different tissues,the soyasapogenol content of hypocotyl is the highest and the lowest in seed coat.2.HPLC/ESI-MS/MS method was uesed to determine the content of soyasaponins in soybean seeds of Wuxiang small black bean at different developmental stages,which containing high content of soyasaponins.And the same method was also used to analyze Jinyi 30 with low content of soyasaponins.The results showed the content of total soyasaponins and all kinds of soyasaponin have a trend of increasing first and then decreasing in different developmental stages.The total soyasaponins content reached the maximum during 32-40 day after flowering,mainly consisted of group A and B,while the group E is the least.There is significant difference in the composition of group A Soyasaponins in Wuxiang small black bean and Jinyi 30,while soyasaponin Aa is the main component in Wuxiang small black bean,and soyasaponin Ab enrich in Jinyi 30.The main component of group DDMP soyasaponins is soyasaponin(3g in the two cultivars.3.293 new genes were obtained from the transcriptome sequencing of the Wuxiang black bean and Jinyi 32 days after flowering,in which 259 genes were annotated.1725 differentially expressed genes were obtained,in which 1684 genes were annotated.The number of SNP was 70896 and 33243,respectively,compared with soybean genome in Wuxiang small black bean and Jinyi 30.The variable splicing type is mainly TSS and TTS.There are three differentially expressed genes involved in the synthesis of Soyasaponins,which are GmHMGRl,?S and UGT73B5,respectively.4.The GmHMGR1 gene was cloned in Wuxiang small black bean and Jinyi 30,and analyzed by bioinformatics.The results showed that the soybean GmHMGR1 gene consisted of four exons and three introns.The length of genomic sequence is 4270 bp,and length of cDNA sequence is 1632 bp,which encoded 543 amino acids.The GmHMGR1 gene encoding protein of Wuxiang small black bean and Jinyi 30 only in the 152 position from D(aspartic acid)into N(asparagine).This site has no significant effect on protein structure.GmHMGR1 gene encoding protein has close genetic relationship with Phaseolus vulgaris,distant relationship with Prunus persica.The amino acid sequence of the cloned GmHMGR1 gene has the conserved domain of HMGR gene family,which contains HMG-CoA binding site TTEGCLVA and two NADP(H)binding sites GTVGGGT and DAMGMNM.5.The expression patterns of GmHMGR1 gene in different tissues and soybean seed was analyzed,the expression of ?AS and UGT73B5 gene in soybean seed was analyzed.The results showed that GmHMGRl gene is expressed in different tissues,and the expression level is different in different tissues.The expression of GmHMGR1 gene in different period grain presents the trend that first increased and then decreased,and the content of soyasaponins has almost the same change trend.The expression of PAS and UGT73B5 genes also show the trend of decreasing after the first increase.Finally,It was proved that GmHMGR1,?AS and UGT73B5 genes are the key enzymes of soyasaponins biosynthesis.