Optimization Of Soybean Embryonic Tip Genetic Transformation System Of Jilin 35 And Gene GmMYB12B2 Genetic Transformation

Soybean（Glycine max（L.）Merr）is not only an important economic crop and oil crop,but also rich in flavonoids such as isoflavones,which are also attracting attention in medicine and health care.With the development of molecular biology,it has become possible to use genetic engineering to improve the quality of soybeans.This study optimized the genetic transformation system of soybean embryo tips,constructed a highly efficient overexpression vector,transferred GmMYB12B2gene into soybean,and completed the detection of transgenic lines.The overexpression vectors pCHF-3300-GmMYB12B2（35S promoter）and pBA-002-GmMYB12B2（soybean seed-specific promoter ProGmOLE8）were first constructed.Taking Jilin 35 soybean apex as explant,the method of scratching the embryo tip,the mode and time of Agrobacterium infection,the concentration of Basta screening agent and other factors that may affect the transformation of Agrobacterium were adjusted and optimized.The results showed that the regeneration rate of the longitudinally scratched plants was the highest in the three kinds of scratches of Jilin35.The best time for vacuum infection was 15 min,the best infestation time was 5 h,and the shaker was 5 h.The regeneration rate of the infected plants after 5h in the shaking bed was higher than that of the vacuum for 15 minutes.In the bud induction and stem regeneration stage,the screening agent Basta concentration of 1.8 mg/L was the best.Based on these factors,the optimized system reduces the occurrence of false positives,and the embryonic explants have high regenerative capacity,which shortens the culture period to some extent,indicating that the optimized genetic transformation system is feasible.Each independent transformation event was detected and verified by PCR,PAT protein strip,Southern blot and qRT-PCR,respectively.PCR and PAT protein strips initially proved that 3 strains（T₀-4,T₀-5,T₀-7）in the T₀ generation were positive for GmMYB12B2 overexpressing lines;The T₁generation strains were further verified by Southern blot hybridization,and the results showed that 3 strains（T₁-4,T₁-5,T₁-7）were positive;The expression of GmMYB12B2 in T₁ transgenic lines was detected by qRT-PCR with Jilin 35 as control.The expression of GmMYB12B2 in T₁ transgenic lines was significantly increased compared with wild type,and the expression of key enzyme genes in isoflavone metabolic pathway was detected.The expression levels of DFR and FLS were significantly different from those of wild type,and the expression level of CHS was extremely significant.