Construction of RNA interference vector of Polygonum cuspidatum chalcone synthase gene and its genetic transformation / 中草药

ABSTRACT

Objective: To construct the RNAi expression vector of Polygonum cuspidatum chalcone synthase (PcCHS1) gene, and to obtain the transgenic plants in which PcCHS1 expression was down-regulated. Methods: According to known sequence (EF090604) of PcCHS1 gene in GenBank, right primers were designed and the conserved sequence was cloned. The conserved fragment (574 bp) targeting at PcCHS1 gene was inserted into the expression vector pYLRNAi in both forward and reverse directions, and RNA interference (RNAi) expression vector pYLRNAi-PcCHS1 was constructed. Using the method of Agrobacterium-mediated transformation, the expression vector was used to transform the shoot tips of P. cuspidatum, and transgenic plants were obtained. The expression of PcCHS1 was confirmed by Northern blotting and the accumulation of polydatin was detected by HPLC. Results: RNAi expression vector of PcCHS1gene was constructed successfully, and five transgenic plants were obtained. Northern blotting analyses indicated that the expression levels of PcCHS1 were significantly down-regulated in the transgenic plants. Polydatin concentration in the transgenic plants was up to 3.8 times higher than that in non-transformed control plants. Conclusion: Transgenic P. cuspidatum plants with down-regulated expression of PcCHS1 gene were obtained successfully. The content of polydatin in the transgenic P. cuspidatum was significantly increased by RNAi against PcCHS1. This work might establish an experimental basis for the effective application of PcCHS1 in improving polydatin accumulation in P. cuspidatum.