Cloning Of The Arabidopsis Stachyose Synthase Gene And Its Transformation Of Maize

Seed vigor is critical for crop production.Raffinose family oligosaccharides(RFOs)plays an important role in seed vigor and plant abiotic stress tolerance.The high degree polymerized RFO(HDP-RFOs)contribute more than raffinose to the seed vigor in Arabidopsis.However,HDP-RFOs was not found in maize seeds.To investigate whether HDP-RFOs would increase the maize seed vigor,the Arabidopsis stachyose synthase gene(At STS)was cloned and transformed into maize immature embryo.Total 7 transgenic maize R0 plants were obtained.The main results are shown as following:Arabidopsis stachyose synthase gene(At STS,At4g01970)was successfully cloned from the seeds of wild type(Columbia-0)using RT-PCR method.The bacterial expression vector of At STS was constructed and expressed in bacteria.The bacterial expressed At STS was confirmed to have stachyose synthetic activity as determined by in vitro enzyme activity assay.The purified bacterial expressed At STS was applied for rabbit immunization.After immunization for 4 times,polyclonal antibody titer detection was performed.The polyclonal antibody was able to detect 10 ng of bacterial expressed At STS protein.The stachyose was accumulated much higher in At STS overexpressing Arabidopsis plants than that wild type plants.The maize expression vector of At STS was constructed and transformed into maize inbred line A188 immature embryo using agrobacterium tumuficiens method.Total of 7 R0 maize transgenic plants were obtained.Further analysis showed that At STS gene was transcribed and translated using RT-PCR or western blot analysis.HPLC analysis shows that stachyose was accumulated much higher in transgenic plant leaves compared with that of control.In summary,we have successfully obtained the transgenic maize plants overexpressing At STS gene.This study lays the foundation of future research work for improvement of maize seed vigor.