Abstract:

Amylose from potato starch is of great advantage for applications in many fields because of its higher degree of polymerization and lower gelling temperature compared with cereal starch. But there is no natural mutant of high-amylose potato. RNAi technique is efficient and specific for plant gene silence but traditional RNAi vector is laborious to prepare. To design an easily-prepared RNAi vector and to develop transgenic high-amylose potato, PCR technique was employed to amplify the nos terminator, the tobacco axi1 and the tobacco ubi.u4 promoter, and to sub-clone a fused fragment SIII which is partially homologous to potato Sbe1 and to Sbe2. Then, a newly designed vector pCUSNI containing “ubi.u4 promotor–antisense SIII–antisense nos terminator–axi1 gene intron–sense nos terminator” was generated and transformed into potato varieties Longshu 3, Gannongshu 2, and Atlantic by Agrobacterium-mediated transformation. Sixteen transgenic potato plants were obtained, and the amylose content in 14 of them increased significantly, which ranged from 53.80% to 85.33% of total starch. But with the increase of amylose content, starch content decreased in transgenic potato plants. Results of semi-quantitative RT-PCR indicated that the accumulation of mRNAs for Sbe1 and Sbe2 was not detectable in transgenic plants with amylose content higher than 80%, indicating that the novel RNAi vector pCUSNI is highly efficient for the simultaneous silence of Sbe1 and Sbe2 in potato. The generation of the RNAi vector pCUSNI makes it much easier to prepare RNAi vectors targeting to other plant genes. The only thing is to insert a fragment of the target gene or the target genes in restriction sites between BamH I and Xba I in vector pCUSNI to replace SIII and there is no need to construct an inverted repeat of target gene.

Key words: RNAi, nos terminator, Inverted repeat, high-amylose potato