Molecular Mechanism Of Soybean MYB Transcription Factors GmMYB68 And GmMYB3a Regulating Isoflavone Synthesis

Soybean is an important food crop and has received widespread attention due to its high content of isoflavones.Isoflavones are important secondary metabolites of plants and have a high content in soybeans.Soybean isoflavones,as health products,have good prospects and application value in areas such as antioxidant,antibacterial,and estrogenic effects,especially in antioxidant,prevention,and treatment of cardiovascular diseases.At present,there are few MYB transcription factors involved in regulating soybean isoflavone synthesis.Mining and identifying genes related to regulating isoflavone accumulation can provide genetic resources for the cultivation of new soybean strains with high isoflavone content.Our laboratory screened and cloned MYB transcription factors Gm MYB68 and Gm MYB3a from the expression profiles of soybean embryos at different stages in the early stage.Through soybean database and chromosome mapping analysis,it was found that molecular markers near the two transcription factors were related to isoflavone content.Using Agrobacterium mediated soybean cotyledon node genetic transformation method,soybean variety Williams82was genetically transformed and Gm MYB68 and Gm MYB3a transgenic soybeans were successfully obtained.On this basis,further studies were carried out in this paper,including sequence and location analysis,screening and identification of transgenic strains,determination of target products（target metabolites）,transcriptome sequencing,investigation of agronomic traits,yeast one hybrid and yeast two hybrid experiments,which partially analyzed the molecular mechanism of Gm MYB68 and Gm MYB3a involved in regulating isoflavone synthesis.The main research findings are as follows:1.The Gm MYB68 and Gm MYB3a genes were constructed into an evolutionary tree with identified and isoflavone related MYB transcription factors.Sequence analysis showed that Gm MYB68 and Gm MYB3a had high homology with identified and isoflavone related MYB transcription factors.Construction of fusion expression vectors p CAMBIA3301-Gm MYB68-2*flag GFP and p CAMBIA3301-Gm MYB3a-2*flag GFP.In tobacco,subcellular localization results showed that both genes were mainly located in the nucleus.2.Gm MYB68 and Gm MYB3a transgenic lines were identified using ammonium phosphate coating,PCR,and strip detection methods.Finally,four stable T₃ generation Gm MYB68 overexpression lines and four Gm MYB3a overexpression lines were obtained.3.High performance liquid chromatography was used to measure the isoflavone content of genetically modified soybeans for three consecutive years.The results showed the same trend for three consecutive years,indicating that overexpression of Gm MYB68 and Gm MYB3a genes can increase the isoflavone content of soybeans.4.Transcriptome sequencing was performed on Gm MYB68 and Gm MYB3a overexpression strains and control strains,and a molecular network diagram of MYB transcription factors involved in regulating isoflavone synthesis was constructed,as well as a thermographic analysis of key enzyme genes in isoflavone synthesis pathway.The results indicate that Gm MYB68 may regulate isoflavone content by regulating the Gm CHS7,Gm CHS8,and Gm CHR6 genes in the isoflavone synthesis pathway;Gm MYB3a may regulate isoflavone content by regulating the Gm CHS7,Gm CHS8,Gm CHI1B2,Gm CYP98A2,Gm CYP93A1,Gm IF7GT,Gm IF7Ma T,Gm IFS1,Gm IFS2,and Gm HIDH genes in the isoflavone synthesis pathway.5.Three consecutive years of agronomic trait investigation were conducted on transgenic soybean plants after harvest,and the same results were presented for three consecutive years,indicating that the plant height,bottom pod height,number of main stem nodes,and effective branch number of transgenic plants are similar to those of the wild type.6.By constructing a yeast single hybrid vector and conducting yeast single hybrid experiments,the results showed that the Gm MYB68 and Gm MYB3a genes may participate in the accumulation of isoflavones by regulating the expression of Gm CHS8and Gm CHS7 genes.7.The yeast self activation results showed that both Gm MYB68 and Gm MYB3a exhibited self activation activity.By using yeast two hybrid technology,a soybean yeast nuclear library was screened to obtain 6 candidate proteins that may interact with Gm MYB68 and 6 candidate proteins that may interact with Gm MYB3a.Further yeast two hybrid one-on-one validation results indicate that Gm MYB68 may interact with Gm YABBY11,Gm SBT1.4,Gm KTI1,Gm E2 22,Gm RD21a,and Gmdna J proteins,while Gm MYB3a may interact with Gm RPS6,Gm COL20,Gm PAA1,Gm LAX3,Gm SBT1.4,and Gm MAPK1 proteins to form complexes and participate in the accumulation of isoflavones.