Identification And Analysis Of Soybean GmMYB12B2 Transgenic Lines

Soybean [Glycine max(L.)Merr] is rich in unsaturated fatty acids,protein,soy saponin,soybean isoflavones and other nutrients,so it is also called "plant meat" and occupies an important position in the agricultural economy.Soy isoflavones belong to flavonoids and are the products of secondary metabolism during soybean development.As a kind of phytoestrogens,it has rich nutritional value.Soy isoflavones can affect protein synthesis,growth factor activity and hormone secretion.Studies have found that its pharmacological effects are rich,such as antioxidant,prevention and treatment of cardiovascular disease,menopausal syndrome,osteoporosis,etc.,and it is a potential anti-cancer substance currently studied in the medical field.Therefore,the selection of high-isoflavone soybean varieties has positive significance for medical health and economic development.Studies have shown that the content of soybean isoflavones is not only determined by related enzyme genes in the process of synthesis and decomposition,but also regulated by MYB transcription factors.MYB transcription factor is the most diverse type of transcription factor.At present,studies have confirmed that MYB transcription factor participates in the regulation of the secondary metabolic pathway of phenylpropane in plants,and the flavonoid metabolic pathway is one of its branches.Studies have shown that MYB transcription factors can positively regulate the accumulation of soybean isoflavones,but studies have also confirmed that the overexpression of GmMYB39 gene can inhibit the synthesis of soybean isoflavones.MYB transcription factors have a wide range of biological functions,and their functions involve plant growth and development,adversity stress,and plant secondary metabolism.These previous research results laid a solid foundation for the research of GmMYB12B2 gene.Based on the original transgenic plants in the laboratory,this study completed the identification of GmMYB12B2 transgenic lines at the transcription level and translation level using molecular biology techniques such as Elisa,Western blot,and Southern blot,and completed the survey of agronomic traits of GmMYB12B2 transgenic plants Analysis;The expression level of GmMYB12B2 transgenic plants was analyzed and the isoflavone content of mature soybean seeds was determined.The main experimental results are as follows:1.Southern blot experiment results show that the herbicide resistance gene has been integrated into the soybean genome and inserted as a single copy;Western blot confirmed that both the target protein and herbicide resistance protein have been stably expressed in soybean;the ELISA results showed that Compared with the wild type,the target protein expression levels of the transgenic lines have reached extremely significant differences,among which MYB-1 and MYB-2 reached more than 5 times that of the non-transgenic lines,and MYB-3 reached 3 times that of the non-transgenic lines.the above.2.The results of qRT-PCR showed that the expression level of this gene was highest in soybean roots,followed by higher expression levels in embryos,and lower expression levels in stems and leaves;the same plant expressed roughly in the early stages of embryo development It increased as the embryo matured,but the expression level in the 30-day embryo declined,and the expression level in the 40-day embryo rose again.3.The detection and analysis results of the target product showed that the isoflavone content of MYB-1 and MYB-2 transgenic plants was significantly higher than that of the wild type,4.07mg/g and 4.75mg/g,respectively.Among them,the MYB-2 strain Compared with the wild type,the increase was about 1.82 times,mainly because the increase of daidzein and genistin were more significant,which increased by 1.77 times and 1.90 times respectively.It shows that overexpression of GmMYB12B2 gene in soybeans helps to increase the content of soybean isoflavones.4.The comparative analysis of agronomic characteristics of GmMYB12B2 transgenic soybeans and recipient control soybean materials showed that the MYB-1 strain had significant differences in plant height and bottom pod height compared with the control,and MYB-2 was compared with the control in plant height.Compared with the control,there are significant differences,and the other agronomic performances of the control and the transgenic plants are not significantly different.