用RNA干扰技术创造高直链淀粉马铃薯材料

Using RNAi Technology to Produce High-Amylose Potato Plants

【目的】创造块茎高直链淀粉含量的转基因马铃薯材料。【方法】采用RT-PCR技术分别克隆了马铃薯Sbe1基因CDS内300bp的片段SⅠ和Sbe2基因CDS内410bp的片段SⅡ,并将SⅠ和SⅡ顺序连接得到融合片段SⅢ;以载体pHANNIBAL和pART27为基础,构建具有SⅢ反向重复结构的植物表达载体pRNAiⅢ;采用农杆菌介导法转化马铃薯优良品种陇薯3号、甘农薯2号和大西洋。【结果】获得了融合片段SⅢ,构建了以Sbe1基因和Sbe2基因为靶标的RNA干扰载体pRNAiⅢ,通过农杆菌介导法获得了24个转基因株系,其中21个转基因株系试管薯的淀粉粒形态发生明显变化,表观直链淀粉含量介于59.31%～87.14%,比受体材料平均高出3.2倍。RT-PCR分析表明,在8个直链淀粉含量超过80%的转基因株系中检测不到Sbe1和Sbe2基因mRNA的积累。【结论】采用RNAi技术通过沉默内源Sbe1和Sbe2,可获得高直链淀粉含量的马铃薯材料。

【Objective】To develop transgenic potato （Solanum tuberosum L.） plants with high-amylose starch in its tubers. 【Method】RT-PCR was employed to clone a 300 bp fragment SⅠfrom the CDS of Sbe 1 and a 410 bp fragment SⅡ from the CDS of Sbe 2. Then the two cloned sequences were ligated in tandem to get a fused DNA sequence SⅢ. Plant expression vector pRNAiⅢ with inverted repeats of S Ⅲ was constructed based on the vectors pHANNIBAL and pART27. Finally,the inverted repeat construct was transformed to elite potato cultivars ‘Longshu 3’, ‘Gannongshu 2’ and ‘Atlantic’ by Agrobacterium- mediated transformation. 【Result】A fused fragment SⅢ was got and its inverted repeats expression vector pRNAiⅢ was constructed. Twenty-four transgenic potato lines were obtained. Starch of in vitro tuber stained with iodine and visualized under microscope evidenced that 21 out of the 24 transgenic lines showed a phenotypic change of starch granule structure. The determination of amylose content showed that starch from these 21 lines had an apparent amylose content of 59.31%-87.14% which was 3.2 times higher than their parental lines in mean. Result of semi-quantitive RT-PCR indicated that the accumulation of mRNAs derived from Sbe1 and Sbe2 were not detectable in 8 tansgenic lines whose amylose content was higher than 80%. 【Conclusion】RNAi is an efficient gene silencing method and can be used effectively in the production of high-amylose potato by silencing endogenesis genes Sbe1 and Sbe2.