Study On Transformation Of Arabidopsis Thaliana AGPase Gene In Maize

Corn, the "King of grain", is rich in nutrition. Corn occupies an important position in theworld’s agricultural productions, it is an universal raw materials of food, feed and industrial.Corn starch, the main nutrients in maize endosperm, is not only the main components of thehuman food, but also an important industrial raw material widely used in medicine, paper,adhesives, textiles, brewing, biodegradable plastics and other industries. In normal corn, only68%proportion does crude starch content accounts for the dry weight of maize grain, thestarch content can no longer meet the needs of social progress. Therefore, the application ofgenetic engineering techniques for quality improvement of ordinary corn, starch content andquality, and to provide a new type of starch raw materials has become particularly important.In starch biosynthesis, AGPase is the rate-limiting enzyme of the entire process, the reactionwhich it catalyzes is an important regulate loci in starch accumulation process. In this test, twoplant expression vectors, pCAMBIA3301-RP5-AtAGPS and AtAGPS-Hord-pCAMBIA3301-RP5-AtAGPL, were constructed based on pCAMBIA3301vector, with a rice endosperm specificpromoter RP5and a barley endosperm-specific promoter D-hordein, and two genes AtAGPL andAtAGPS who encoding the AGPase’s large and small subunit in Arabidopsis.Through Agrobacterium-mediated method, we transferred AtAGPS into callus ofmaize inbred line YW9801successfully, and obtain67regenerated plants at present.We could take them to some transgenic test at next step.We used the upland selfing line of corn Chang7-2as target materials, andtransformed the expression vector pCAMBIA3301-RP5-AtAGPS into the corninternode of this varietie via Agrobactetium-mediated.85regenerated plants wereobtained,3of which were verified to be transgenic plants after two rounds of PCRdetection based on primers of Bar and the target genes. Then, this3transgenic plants of Chang7-2were also obtained by Bar gene test strip examination and Southernblotting hybridization analysis, proof AtAGPS gene had into genomic sequence ofcorn, the transformation frequency was about to3.5%.Plant expression vector pCAMBIA3301-RP5-AtAGPL were transformed intomaize inbred lines: YW9801，YW9812，PH6wc，PH4cv，Zheng58and Chang7-2through pollen tube pathway.74ears were transformed and22067T0seedsgenerated. After Basta resistance screening and three rounds of PCR detection basedon primers of Bar, promoter and the foreign genes,21T0transgenic plants wereobtained, the transformation frequency was about to0.1%. In the second year,15ears were transformed and2767T1seeds generated. And determination of the starchcontent of the T1seeds, compared with control, the starch content of transgenic seedshave improved0.2％~0.8％.The same methods of detection were used in the T1generations to verify the transgenic plants. We obtain1T1transgenic plants, thedetection rate was0.04%. At present, we have obtained the transgenic seeds of T2generations.