【作者】 李桂民；

【导师】 刘立侠；

【作者基本信息】 东北师范大学 ， 生理学， 2005， 硕士

【摘要】 基因工程是作物育种的重要手段,通过转基因技术可以改良植物的性状和其产品的营养价值。近年来,双链RNA介导的转录后基因沉默技术在油料作物脂肪酸组成的改良方面的研究获得了突破性进展。其原理是,利用反向重复序列的基因沉默构建并转化植物,该构建可在体内转录为mRNA后,可以自我互补而形成发卡式RNA(hairpinRNA,hpRNA),而hpRNA所含有的双链RNA结构可导致特异基因编码的mRNA降解。 油酸是一种单不饱和脂肪酸,能降低有害胆固醇(低密度脂蛋白)但保持有益胆固醇(高密度脂蛋白)的水平,从而有效预防冠心病等心血管疾病的发生。高油酸植物油是健康的、稳定的和高品质的食用油,因此提高油酸的相对含量,增加油酸/亚油酸(O/L)的比值,已成为目前油料作物品质改良育种的重要内容。 ahFAD2B是编码花生微粒体ω6-脂肪酸脱氢酶的主要基因,本研究利用hpRNA介导的基因沉默技术抑制该基因的表达,从而提高油酸的含量,改善花生油脂肪酸的组成。根据Gene Bank公开发表的ahFAD2B基因序列设计特异引物进行PCR,将扩增出的反义臂和正义臂连接成反向重复序列,并以大豆凝集素基因的启动子来启动其转录。将该构建通过农杆菌介导转化花生,经过组织培养和继代筛选,获得转基因植株。对转基因植株进行PCR检测,结果表明目的基因已整合到花生基因组中。对T1代种子进行脂肪酸分析,结果表明种子油酸含量由原来的38%提高到77%,而亚油酸的含量由36%降低到9%。这种改良的高油酸花生油含77%的油酸和12%的饱和脂肪酸,具有较高的氧化稳定性,是有益于健康的高品质食用油。本研究所获得的高油酸花生材料为进一步选育遗传性稳定和性状优良的高油酸花生品系或品种奠定了基础。更多还原

【Abstract】 Gene technology has provided plant breeders with powerful new tools for manipulating the composition of plant products to improve their nutritional value and functional properties. Double strands RNA produced post transcriptional gene silencing (PTGS) technique is being used to alter the relative proportion of the major fatty acids present in oil plants. Specifically designed genetic constructs resulted in gene silencing, such as inverted repeats that encode RNA having regions of self-complementarity, can reliably generate hairpin RNA (hpRNA) transcripts that invoke sequence-specific RNA degradation.The monounsaturated oleic acid could contribute to lowering the onset of coronary heart disease (CHD) by reducing blood low density lipoprotein (LDL) cholesterol and not change high density lipoprotein(HDL) cholesterol levels. Vegetable oil with elevated levels of oleic acid provides desirable properties related to both health benefits and stability characteristics. Therefore, one of the major goals for oilseed research is to improve the nutritive value of oilseeds by increasing the ratio of oleic acid to linoleic acid.In this study, we have employed hpRNA-mediated gene-silencing techniques to down-regulate the expression of key fatty acid desaturease gene of peanut, ahFAD2B, encoding microsomal ω6-desaturease and consequently modify the fatty acid composition of peanut oil (with high oleic oil content). The specific primers were designed according to the published sequence of ahFAD2B gene. The fragments of sense and antisense arms were amplified and ligated into an inverted repeat (IR) construct, which was driven by the soybean (Glycine max) lectin promoter. Hairpin RNA-encoding gene constructs targeted against ahFAD2B were transformed into peanut (Arachis hypogaea) by an Agrobacterium tumefacien-mediated method. Identification of transgenic plants by PCR revealed that the constructs had integrated in the plant genome. Fatty acid analysis of T1 seeds showed that the resulting down-regulation of the ahFAD2 gene substantially increased oleic acid content from the normal levels of 38% up to as high as 77%, and linoleic acid was significantly lowered from 36% to 9%. The modified high oleic peanut oil containing predominantly oleic acid (77%) and saturated acid (12%) is high quality edible oil with the stable characters and health benefit. This special peanut line obtained in this study may provide a basic material for genetic breeding of high oleic acid peanut with the stable genetic characters and high quality.更多还原