Transforming Of ADP Glucose Pyrophosphoryiase Gene And Molecular Detecting In Maize

Maize is one of the most important crops in the world and has various direct and indirect applications in food, feed, and economy. Maize starch is one of the most important plant products, is the major component of maize, maize starch is synthesized in the maize seed endosperm. Maize starch has been used to product more than 2500 kinds of industrial products and plays an important role in food and chemical industries. Therefore, increasing the starch content of corn has important economic and practical significance for increasing raw material of industrial products. ADP-glucose pyrophosphorylase plays a key role in regulating starch biosynthesis in cereal seeds, is the major rate-limiting enzyme of starch synthesis in maize endosperm and plays an essential role in the development of grain and corn starch synthesis. It is possible to improve the heat stability of AGPase and improve AGPase activity, reduce sensitivity to inhibition of inorganic phosphorus, increased sensitivity of 3-PGA activation and promote starch synthesis, increase the amount of maize starch in the endosperm, thereby increasing the yield of maize by the artificially modification of AGPase gene.In this study, arificially modified Sh2 gene and BTTP-Sh2 gene were cloned into prokaryotic expression vector pGEX-4T-1, these protein expression in E.coli BL21(DE3) was confimed by SDS-PAGE analysis. The results show that Sh2 gene was highly expressed in E.coli BL21(DE3), BTTP-Sh2 gene also was expressed, but the amount of protein expression is less.Maize is one kind of monocotyledons, it is more difficult to gain transgenic maize plants by using Agrobacterium-mediated, because maize cells is not a natural host of Agrobacterium and not susceptible to the intrusion of agrobacterium. It provides a fast and effective method of genetic transformation when gene gun technology occured for transforming gene. In this study,artificial modification of AGPase gene construct transformed into a plant expression vector pCAMBIA3300, the use of maize 27 kD zein gene promoter driving AGPase gene expression,choose another Bar gene as a selectable marker gene. AGPase gene was introduced into corn hybrids HiⅡA × B and HiⅡB callus by particle bombardment, and obtained herbicide resistantcallus by bialaphos resistance screening, After screening, regeneration, and get the transgenic plants. The transgenic plants were preliminary identified by PCR and by immune test paper.It is initially concluded that AGPase gene was integrated into the corn genome by modified molecular biology research,AGPase gene successful conversion provided new germplasm for the study of the transgene expression, starch synthesis, grain yield and other agronomic traits of the transgenic homozygous plants and laid the foundation for high-starch corn breeding.