【作者】 宋凯；

【导师】 刘立侠；

【作者基本信息】 东北师范大学 ， 植物学， 2005， 硕士

【摘要】 油酸是重要的植物脂肪酸,它性质稳定、不易氧化并且有益于健康。研究表明,油酸能降低有害胆固醇(低密度脂蛋白,LDL),保持有益胆固醇(高密度脂蛋白,HDL)的水平,从而减缓动脉粥样硬化,有效预防冠心病等心血管疾病的发生。近来的研究发现油酸在女性乳腺癌方面起着积极的作用。目前,应用传统选育、诱导突变和新兴的转录后基因沉默等技术,在培育高油酸油料作物方面已经取得了许多突破性的进展,特别是近年来应用转录后基因沉默技术调节脂肪酸代谢过程中酶活性获得的成功。但是转录后基因沉默技术的反义和共抑制手段难以保证抑制的效率和预期结果,往往需要大量的转化材料来挑选所需要的理想表达性状。反向重复 DNA 序列构建诱导的转录后基因沉默技术解决了这个问题,该构建可在体内转录为 mRNA,这种 mRNA 可以自我互补形成发卡 RNA(hpRNA),而 hpRNA 可导致特异基因编码的 mRNA 降解。本研究利用 hpRNA 介导的基因沉默技术来抑制大豆种子中一个重要的脂肪酸脱氢酶——编码油酰磷脂酰胆碱ω-6 脱氢酶基因 gmFAD2-1 的表达,从而提高油酸的含量,改善大豆油脂肪酸的组成。采用大豆凝集素基因的启动子来启动一段可以编码形成发卡 RNA 的反向重复序列 DNA,再通过根癌农杆菌转化大豆,组织培养和继代筛选,获得转基因大豆材料。这样在大豆凝集素基因的启动子的作用下,gmFAD2-1 的反向重复序列结构在大豆种子中的表达可以特异性的沉默种子中的油酸脱氢酶 gmFAD2-1 基因。PCR 检测转基因植株,结果表明目的片断已整合到植物基因组中。更多还原

【Abstract】 As a major component of plant fatty acid, oleic acid with the oxidative stability, flavor characteristics and physical properties contributes significantly to decrease the risk of cardiovascular disease due to the effect on lessening low-density lipoprotein cholesterol in the bloodstream. Recent researches showed that oleic acid could also decline the risk of the breast cancer development in women. There is a growing trend of producing high oleic crops using molecular techniques such as conventional selection, induced mutation and, more recently, post-transcriptional gene silencing (PTGS). In particular, PTGS has been used to down- regulate the activity of the desaturase enzymes that control the synthesis of the major seed fatty acids. However, the antisense and cosuppression strategies used in fatty acid modification have variable and unpredictable effectiveness and generally require the selection from large populations of transgenic plants to obtain an acceptable number of lines exhibiting sufficient degrees of target gene suppression. Recently, a much more effective methods of silencing plant genes have been developed based on the discovery that PTGS can be invoked at very high frequency using the inverted-repeat DNA constructs. Specifically designed genetic inverted repeats constructs resulted in gene silencing. This inverted repeats that encode RNA have regions of self-complementarity and can reliably generate hairpin RNA (hpRNA) transcripts that invoke sequence-specific RNA degradation. We have genetically modified the fatty acid composition of soybean (Glycine max) seed oil using the technique of hairpin RNA-mediated gene silencing. By this technique, the key fatty acid desaturease gene( gmFAD2-1) encoding oledyl-phosphatidylcholineω-6 desaturease was down-regulated resulting in the consequently modification of the fatty acid composition of peanut oil with high oleic oil content. Hairpin RNA-encoding gene constructs (HP) was driven by the seed-specific soybean lectin promoter and tranfered into soybean by Agrobacterium tumefacien-mediated method targetting against gmFAD2-1. Identification of transgenic plants by PCR revealed that the constructs had integrated in the plant genome.更多还原