Construction Of RNAi Expression Vector For Potato Sbe A And Sbe B Gene And Its Transformation Of Potato

Potato (Solanum tuberosum L.) starch is one of the most important plant raw materials for both food and industrial applications. Especially, High-amylose starch is in great demand by the starch industry for its unique physicochemical properties. However, so far the amylose content of the potato cultivation species accounts for 17% of total starch content. Therefore cultivate high-amylose potato species will extend the potato application and value.In the past, the search for high-amylose potato was mainly achieved by extensive breeding programs and the characterization of mutant varieties.Potato has been very difficult to work efficiently using mutational breeding due to the tetraploid nature and hexaploidity.RNA interference technique (RNAi) is a powerful tool to crop genetic improvement. In order to make sbe A and sbe B gene post-transcriptional gene silencing, we do some work:The sbe A gene was amplified by RT-PCR from potato tubers'mRNA. The nucleotide sequence of the clone was confirmed by sequencing, the homology between this 415 bp fragment and the corresponding reported sequence is 99%. The sbe B gene was amplified by RT-PCR from potato tubers mRNA. The nucleotide sequence of the clone was confirmed by sequencing, the homology between this 302 bp fragment and the corresponding reported sequence is 99%. Through sub-cloning, we put this two fragment together.The drepp 1 gene was amplified from tobacco DNA. The nucleotide sequence of the clone was confirmed by sequencing, the homology between this 300 bp fragment and the corresponding reported sequence is 94%. As the partition area, this 300 bp fragment will increase the efficiency of post-transcriptional gene silencing. Then by introducing sbe A and sbe B gene into plasmid pB13, we constructed the sbe-RNAi plant expression vector with CaMV 35S promoter, and the plant expression vector was integrated into agrobaterium LBA4404.The potato cultivar'Longshu 3','Favorita'and'Shapody'were used as plant material to establish the efficient regeneration system by choosing different mediums. At the same time, an efficient transformation system was developed,including the pre-culture of explants, the time of transformation and co-culture. The results indicated that: The optimal duration of pre-culture was 2 days that would improve the rate of stem cutting transformation. The duration of 2 days co-culture might improve the rate of stem cutting transformation. The duration of 1 days co-culture would improve the rate of potato slice transformation.