Detection And Generation Of Transgenic Maize(Zea May L.)Plants Overexpressing High Lysine Protein Gene Sb401

Maize is the most important grain, feed and economy applicable crop, but in the low content of corn lysine, every gram only containing about2.5g lysine, even lysine to man, livestock and poultry is a must, which has caused the utilization rate of the proteins in the corn is very low. Increasing protein and lysine content in corn, promoting the rate of digestion and absorption of man and animal to the corn protein will improve the value of corn.The traditional methods of breeding, to some extent, has promoted the content of lysine, but the potential of the methods is not big and time-consuming. It is hoped that improving the quality of the corn will be realized by the methods of genetic engineering technology with the rapid development of modern molecular biology and biological technology. The Agrobacterium-mediated transgenic method has many advantages over other gene transformation methods, for example, transferring a relatively large segments of DNA, high frequency of single or low copy insertion events, not easy causing rearrangement within molecules or intermolecular, high transformation efficiency, et al. Its transformation efficiency was limited by materials, bacteria liquid concentration, medium composition and many other factors. So establishing a complete and efficient genetic transformation system is very important.In this experiment, the expression vector containing high lysine protein gene sb401cloned from pollen of potatoes and selecting marker gene bar was transferred into embryogenic maize callus of hybrid line Hi ⅡA×B by Agrobacterium tumefactions-mediated transformation approach. The agrobacterium we used was EHA105strains, and the material was embryo callus of Hi ⅡA×B, and the concentration of the agrobacterium suspended was OD600≈0.6～0.8, and the infection time was5min, and the coculturing time was3～4d. The bialaphos concentration was1.5mg/L in the frist selection, and3mg/L in the later selection, total screening3～4times.15transgenic plants were identified by PCR from70regenerated transformed plants obtained by selection for bialaphos resistance. PCR-southern blotting assay for the transgenic plants indicated that high lysine protein gene sb401has been integrated into genome of maize. Bar protein dipsticks detection was positive, so we can infer the target protein was expressed. The positive rate of transgenic maize plants T1was23.2%by PCR and PPT detection method, which proved high lysine protein gene sb401has been stable genetic.