Analysis Of The Reason Of PAL2-3 In Regulating Isoflavone Content In Soybean Seeds

Soybean isoflavone is a secondary metabolite which mainly exists in soybean,It has attracted much attention because of its important functions in human physiological health care and plant itself.Soybean isoflavone synthesis and metabolism pathway is a branch of phenylalanine metabolism pathway,and phenylalanine ammonia lyase?PAL?is the key enzyme and rate-limiting enzyme in phenylalanine metabolism pathway.Previous studies showed that PAL2-3?GLYMA₁3G145000?gene had a regulatory effect on isoflavone content in soybean seeds:isoflavones and three aglycones in soybean grains were increased in T 1 transgenic soybean plants overexpressed by PAL2-3 gene.The contents of isoflavones and three aglycones in soybean grains were significantly decreased in T1 transgenic soybean plants with PAL2-3 gene RNAi interference.In order to analysis of the reason of PAL2-3 in regulating isoflavone content in soybean seeds,the results are as follow:?1?By using Bar gene for PCR detection,the relative expression of PAL2-3 gene was determined by Real-time fluorescence quantitative PCR,three transgenic soybean lines of overexpressing PAL2-3 gene and four transgenic soybean lines interfering with PAL2-3 gene were obtained in T2 generation.The activity of PAL in transgenic lines and wild-type control group was determined.The results showed that the PAL activity of overexpressed PAL2-3 liness was higher than that of wild-type control group,but there was no significant difference between the two groups,the PAL activity of PAL2-3 gene lines interfered by RNAi was lower than that of wild type control group.There is a significant difference between the two.The content of isoflavones in mature grains of transgenic lines and wild type control groups was determined.It was found that there were significant difference in the content of isoflavone between different transgenic lines and wild type control group.The contents of isoflavones and their components in different overexpressing PAL2-3 lines were higher than those in wild type control group.?2?During growth and development?R5,R6,R7?,the real-time fluorescence quantitative PCR analysis of isoflavone metabolic pathway related enzymes genes in different over-expressed PAL2-3 gene lines,wild-type control group?Dongnong 50?and different RNAi interference PAL2-3 gene lines showed that:the relative expression of CHI1 B gene and F3 H gene decreased with the development of growth period,but the relative expression of other genes increased with the development of growth period.In the isoflavone synthesis and metabolism pathway,th e relative expression levels of related enzymes genes?C4H,4CL1,CHR,CHS1,CHS7,CHS8,CHI1 A,CHI1B,IFS2 and F3H?in grains of wild type control group were compared,The r elative expression of related enzymes genes in the grains of over-expressed PAL2-3 gene lines increased,the relative expression of related enzymes in grains of RNAi interference PAL2-3 gene lines decreased.?3?The analysis of metabolic substrates of isoflavone pathway showed that cinnamic acid,sinapyl alcohol,daidzein,glycitein and genistein were positively regulated by PAL2-3 gene,while p-Coumaric acid was negatively regulated by PAL2-3 gene.In PAL2-3 RNAi interference lines,coumaric acid,sinapyl alcohol,daidzein,glycitein and genistein were positively regulated by PAL2-3 gene.