应用ihpRNA干扰技术创制高支链淀粉马铃薯材料

Prodution of High-Amylopectin Potato Plants by Using ihpRNAi Technology

【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。

[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 eultivar ＇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 GBSS1 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 potatoor pure-amylopectin potato by silencing endogenesis gene GBSS1.