用反义RNA技术创造高直链淀粉玉米材料

 【目的】利用反义RNA技术调控玉米淀粉的生物合成过程,创造高直链淀粉玉米材料。【方法】克隆玉米淀粉分支酶(sbe2a)基因片段,以载体pWGLL为基础,构建高效反义表达载体,通过花粉管通道法将其导入玉米自交系铁7922中。【结果】获得了4株转基因株系,GFP表达检测、PCR扩增和Southern杂交结果表明,目的基因已整合到基因组中,且能够遗传。对4株转基因植株进行RT-PCR和淀粉分支酶活性检测,结果表明转反义sbe2a玉米淀粉分子酶基因的转录受到了明显抑制,淀粉分支酶活性明显低于野生型,相差最多的降低79.4%;直链淀粉含量也发生明显的变化,最高的提高了84.3%,且总淀粉含量与对照之间基本没有差异。【结论】采用反义RNA技术通过沉默内源sbe2a,可获得高直链淀粉含量的玉米材料。

Reducing the Maize Amylopectin Content Through Anti-Sense Manipulation

【Objective】 In order to control the biosynthesis process of maize starch and to develop transgenic maize with high amylase starch content. 【Method】 RT-PCR was employed to clone the maize starch branching enzyme gene sbe2a and high efficient anti-sense expression vector was constructed based on plant expression plasmid pWGLL. Then the vector was introduced into maize inbred line Tie7922 by pollen tube pathway. 【Result】 Four transgenic plants were obtained. PCR amplification and Southern blotting hybridization proved that the sbe2a had integrated into maize genome. RT-PCR and enzyme activity results showed that both mRNA level of sbe2a and activity of starch branching enzyme decreased significantly in the transgenic plants than that in the wild type plants and the maximum decrease level of enzyme activity was 79.4%. However the total starch content had no significant difference between the transgenic plants and the wild type plants, while the content of amylose starch increased by 84.3% compared to the wild type plants. 【Conclusion】 Anti-sense RNA is an efficient gene silencing method and can be used effectively in the production of high-amylose maize by silencing endogenesis gene sbe2a.