应用ihpRNA干扰技术创制高支链淀粉马铃薯材料

【目的】创造块茎高支链淀粉或纯支链淀粉含量的转基因马铃薯材料。【方法】以构建的由Patatin启动子驱动的pBI121g-PgABI为干扰表达载体，采用农杆菌介导法转化马铃薯优良品种甘农薯2号。用PCR、Southern blotting、半定量RT-PCR和实时荧光定量PCR技术检测转基因植株，并对转基因植株的微型薯进行淀粉含量的测定。【结果】通过农杆菌介导法获得10个转基因株系。PCR和Southern杂交结果证明，目的基因已被整合到基因组中，通过半定量RT-PCR分析表明，转基因株系中GBSSI的表达均受到明显抑制，且在6个转基因株系中检测不到mRNA的表达。进一步通过real-time PCR分析表明，转基因株系中GBSSI的mRNA沉默效率为66.27%—93.53%；转基因株系微型薯的淀粉含量也发生明显变化，其支链淀粉含量高达90.16%—98.84%，比对照高出10.31%—20.92%。转基因株系GBSSI的mRNA沉默效率与支链淀粉含量呈显著正相关（r=0.937，P<0.01）。【结论】采用ihpRNAi技术可有效抑制马铃薯块茎中内源GBSSI表达，获得高支链或纯支链淀粉含量的马铃薯材料。

Prodution of High-Amylopectin Potato Plants by Using ihpRNAi Technology

【Objective】 The objective of this study is to develop transgenic potato (Solanum tuberosum L.) plants with high-amylopectin starch in its tubers.【Method】RNA interference expression vector pBI121g-PgABI driven by Patatin was transformed into elite potato cultivar ‘Gannongshu 2’ by Agrobacterium-mediated transformation. The transgenic plants were identified by PCR, Southern-blotting, semi-quantitative RT-PCR and real-time quantitative PCR and the starch content of transgenic potatoes was determined.【Result】Ten transgenic potato lines were confirmed by PCR and Southern blot analysis that the target gene integrated into the plant genomes. Result of semi-quantitative RT-PCR indicated that the accumulation of mRNAs derived from GBSSI was inhibited significantly in all transgenic lines, which were not detectable in 6 tansgenic lines. Result of real-time quantitative PCR showed that the inhibition ratio was 66.27%-93.53%. There were significant changes of starch content in ten transgenic microtubers,of which the amylopectin content was up to 90.16%-98.84%, 10.31%-20.92% higher than the non-transgenic microtuber. A significant correlation was found between inhibition ratio of mRNA and amylopectin content of transgenic potato plants (r=0.937, P＜0.01). 【Conclusion】ihpRNAi technology can be used effectively in the production of high-amylopectin potato or pure-amylopectin potato by silencing endogenesis gene GBSSI.