Cloning Of PPase Gene And Genetic Transformation In Potato (Solanum Tuberosum L.)

Potato tuber dormancy and sprouting is an extremely complicated biologic process, which is affected directly or indirectly by numerous environmental, physiological and genetic factors during potato production and storage periods, but its mechanism is not completely clear until now. Potato tuber dormancy and sprouting is very important to potato cultivation, tuber production and processing. Inorganic pyrophosphatase (PPase) is a kind of hydrolase which takes pyrophosphate (PPi) as substrate. PPi is a co-substrate in several steps of plant sugar metabolism, and the content of PPi can link sucrose synthesis in cytoplasm and starch degradation in plastid, which is an important regulation point of sucrose synthesis. The characteristics of potato tuber dormancy and sprouting could be controlled by regulating the content of PPi under the control of PPase.In the present study, PPase gene was cloned from the leave of potato cultivar Atlantic using reverse transcription-polymerase chain reaction (RT-PCR) technology. The expression vectors of the antisense PPase gene were constructed under the control of the constitutive promoter CaMV 35S, tuber-specific expression promoter CIPP and the low temperature-induced promoter rd29A, respectively, and further were introduced into Agrobacterium tumefaciens strain LBA4404. The transgenic potato plants were obtained by Agrobacterium-mediated transformation. The main results as following:1. Total RNA was extracted from leaves of potato cultivar Atlantic. The cDNA of potato PPase gene was amplified using RT-PCR method. The results from sequence analysis showed that the cDNA was 673 bp with 636 bp open reading frame (ORF) encoded a 211-amino acid polypeptide. The GenBank accession number for the PPase gene was EF091820. Homology analysis by blast showed that the PPase cDNA shared 97.62% identity to the published sequence (GenBank accession number Z36894).2. The plant expression vectors pBIAP, pBCAP and pBrAP for the antisense PPase gene, driven by the constitutive promoter CaMV 35S, tuber-specific expression promoter CIPP and low temperature-induced promoter rd29A respectively, were constructed by enzyme digestion and lingation method.3. The putative transgenic potato plants of early-muture cultivar Favorita and late-muture cultivar Gannongshu 2 were obtained by Agrobacterium-mediated transformation method using the plant expression vectors pBIAP, pBCAP and pBrAP. PCR analysis preliminary showed that the PPase gene was transferred to the transgenic potatoes.