Construction Of ShSUT4RNAi Plant Expression Vector And Genetic Transformation

Sugarcane is a high effective photosythesis C4species,mainly grown in the tropical and subtropical regions.Sucrose is major storage substance of sugarcane,and is also the main form of photosynthesis product transporation.Sucrose transport from source organs to sink organs,the process is a high integrity system regulated by multiple organization structures and biochemical pathways. Sucrose transport across the membrane and distribute in sugarcane depend partly on the sucrose transporters located in the plasma membrane. So the sucrose transporter plays an important role in the process of sucrose distribution and accumulation.The sugarcane SUT4gene cDNA fragments(1611bp) has already been cloned in our lab,and we have done part of functional verification.In order to further explore the ShSUT4function,we can inhibit or down regulate the expression of sugarcane SUT4gene through RNAi anti-sense technique,giving different phenotypes of sugarcane.RNAi is an efficient approach to inhibit gene expression,compare to the anti-sense technique; the RNAi effect is more significant.To further verify the ShSUT4function,we inserted a reverted repeat of SUT4into RNAi vector-PTCK303,and constructed a RNAi plant expression vector.Then it was introduced into Agrobacterium Tumefaciens EHA105through freezing and thawing method.It was introduced into sugarcane by Agrobacterium-mediated transformation.20PPT-resistant plants were obtained.After detection of PCR and RT-PCR,3of sugarcane genetically modified plants were obtained.The target gene had been expressed at the transcriptional level in these plants. Then we analysis the positive plants via Gene Expression Pattern and HPLC.The result showed that in transformation plants,ShSUT4expression decreased,sugar content increasedIn conclusion,we presumed that transgenic sugarcane with ShSUT4RI may influence the distribution of sucrose in leaves.All these results can estimate that ShSUT4was correlated with sucrose transport and accumulation in sugarcane,which laid the foundation for intensive study of sucrose accumulation in sugarcane,which also provide the theory basis of cultivating sugarcane varieties with high sugar content.