虎杖查耳酮合酶基因RNAi载体的构建及其遗传转化

目的构建虎杖查耳酮合酶(Pc CHS1)基因的RNA干涉(RNAi)表达载体,获得Pc CHS1表达下调的转基因虎杖植株。方法根据Gen Bank中已知的Pc CHS1基因序列(EF090604),设计相应引物,克隆Pc CHS1基因核心保守序列。以Pc CHS1基因为靶基因,将长度574 bp保守序列片段通过正、反2个方向插入表达载体p YLRNAi中,构建RNAi表达载体p YLRNAi-Pc CHS1。通过根癌农杆菌介导法将其导入虎杖茎尖组织。对获得的转基因植株,利用Northern blotting检测Pc CHS1基因表达水平,并应用HPLC法测定虎杖中白藜芦醇苷的量。结果成功构建Pc CHS1基因RNA干涉表达载体,获得了5株转Pc CHS1基因干涉载体的阳性植株。转基因虎杖Pc CHS1基因表达水平显著下调,并且转基因植株中白藜芦醇苷量均得到显著提高,其中最高量是对照植株的3.8倍(P0.05)。结论成功获得了干涉Pc CHS1基因表达下调的转化植株,抑制Pc CHS1基因的表达显著增加了转基因虎杖中白藜芦醇苷的量,为有效利用该基因提高虎杖白藜芦醇苷量奠定基础。

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

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.