Construction Of RNA Interference Vector Of Potato Sucrose Transporter 2 And Its Genetic Transformation

Potatoes(Solanum tuberosum L.)are one of the world's leading main grain crops,its tubers can not only as food crops,but can be used for industrial production of starch and so on.In photosynthesis of carbohydrates,the phloem sieve to the storage organ transport and accumulation process,the Sucrose transporters are playing a major role.The sucrose transport proteins in the dicotyledonous plants include three specials: SUT1,SUT2 and SUT4,and those main function are regulating the loading,transporting and unloading of the plant photosynthate sucrose.In potatoes,the StSUT1 protein mainly regulates the loading of sucrose.The StSUT4 protein mainly regulates the unloading process of sucrose,and it interacts with the photoperiod to regulate the flowering,stem length and tuber formation of the potato plants,however,the function of the StSUT2 protein is still unknown.According to the research and analysis of higher plants SUT2 in domestic and abroad for many years,its structure is similar to the glucose sensor signals,so it is speculated that SUT2 may be the sucrose signal receptor rather than sucrose transporter.In this study,the potato StSUT2 gene has been constructed and genetically transformed into potatoes,which laid the foundation for the functional analysis of StSUT2 protein.The experiment is mainly divided into two parts: Firstly,construction the RNA interference vector.The Gateway cloning technology can be quickly reorganized into the expression vector,through the recombination of the sequencing,identified as the interference expression vector.Secondly,the potato genetic transformation.The interfering vector is transformed into potato tubers by Agrobacterium tumefaciens,which recombines to produce the transgenic plants containing interfering genes.The main results achieved are as follows:(1)Using the TOPO clone,the amplified interference fragment was integrated into pENTR,and the entry vector pENTR-SUT2.(2)LR recombination was carried out according to Gateway cloning technique.The interference expression vector pSUT2-RNAi was constructed according to the att L and attR loci of the entry vector and the expression vector.The recombinant plasmid pSUT2-RNAi was identified by restriction enzyme digestion and gene sequencing.(3)The interference vector pSUT2-RNAi was transformed into Agrobacterium tumefaciens GV3101 by freeze-thawing method.(4)Transformed into potato tubers named the SHB and L3,and detected by kanamycin screening and the PCR amplification.To determine the interference gene has been successfully transferred into the potato plants.Real-time quantitative polymerase chain reaction(qRT-PCR)showed that the interference gene had been transferred to potato plants and inhibited the expression of the StSUT2 gene.