【作者】 周寅；

【导师】 唐克轩；

【作者基本信息】 复旦大学 ， 遗传学， 2010， 硕士

【摘要】 维生素C,又称抗坏血酸(L-Ascorbate acid,AsA)是植物体内主要的抗氧化物质,参与植物的光合作用和光保护,调控着植物的开花、结实、细胞凋亡、细胞壁代谢、细胞膨大和细胞分裂。人类失去了自主合成AsA的能力,必须从植物中不断获取。通过基因工程技术提高植物AsA含量,改良植物营养品质和抗性是分子育种新的发展方向之一。随着AsA的代谢工程研究的逐渐开展,AsA合成代谢的全部基因已经阐明,系统评价也已完成。本研究通过PCR从拟南芥cDNA中获得AsA合成代谢途径6个催化酶的编码基因,组合成4个双基因载体,在模式植物拟南芥中过表达,比较双基因转化和单基因转化对AsA含量的贡献。同时,也为选择最佳的多基因转化组合提供一些思路。过表达双基因和单基因转基因系,经实时定量PCR分析,目的基因表达量多数都有上调。AsA含量的HPLC分析结果显示,野生型(WT)和转空白质粒植株对照(CK)中AsA含量为35.83±8.64 mg/100g FW和34.84±11.45 mg/100g FW;各GGT-GLDH转基因株系中AsA含量最高为142.53±41.46 mg/100g FW,是相应对照的4.09倍；各GGT转基因对照株系中AsA含量最高126.39±22.53 mg/100g FW,是相应对照的3.63倍；各GLDH转基因对照株系中AsA含量最高61.51±5.03 mg/100g FW,是相应对照的1.77倍；双基因转化株系较单基因转化株系表现略好,但差异并不明显。各GMP-GME转基因株系中AsA含量最高的为44.92±27.17 mg/100g FW,是相应对照的1.29倍；各GGT-GPP转基因株系中AsA含量最高达到142.46±8.80 mg/100g FW,是相应对照的4.09倍；各GDH-GLDH转基因株系中AsA含量最高达到43.10±7.80 mg/100g FW,是相应对照的1.21倍。通过AsA含量的对比和各基因表达量的分析,高AsA含量的株系均有较高的GGT表达,进一步证实了GGT在L-半乳糖途径中是唯一的限速酶,其他基因效果则均不明显。这亦是双基因转化效果不明显的原因。GGT的表达还对其他基因有一定的拉动作用。本研究首次在同一种植物中比较了单基因转化和双基因转化的优缺点,揭示了双基因转化的规律,进一步提示了共转化选取最佳基因组合的一些思路,为制定和优化提高植物AsA含量的遗传工程改造策略提供了初步的实验依据。更多还原

【Abstract】 Vitamin C (ascorbic acid) is a main part of the plant antioxidant system and it plays an important role in photoprotection, flowering, fruit ripening, cell senescence, division, expansion, and cell wall metabolism. However, humans have lost the ability to synthesize AsA, thus have to obtain their AsA regularly from plants. Engineering plant AsA metabolism has now become feasible because of the increased understanding of its biosynthetic pathway and key enzymes. All of the six genes which involved in AsA biosynthetic pathway were cloned by PCR from Arabidopsis thaliana cDNA and evaluated. Four dual-gene plant expression vectors were constructed and transformed into Arabidopsis thaliana for the comparison between single-gene transformation and dual-gene transformation, and also for the selection of the best gene combination.In most of the biothyntic gene transgenic lines, the expression of the target gene was increased on RNA level(real-time PCR). The AsA content was detected using HPLC. The GGT-GLDH transgenic lines increased the AsA content to 4.09-fold compared with the blank transformation(CK); the GGT, GLDH, GMP-GME, GGT-GPP, GDH-GLDH transgenic lines increased the AsA content to 3.63-fold,1.77-fold,1.29-fold,4.09-fold,1.21-fold compared with CK respectively. This result shows that GGT catalyze the only rate-limiting step in L-galactose pathway. The other five genes contribute little to AsA accumulation. This is the reason why dual-gene transformation does not perform markedly better to single-gene transformation. The result also indicates that GGT has the pull-up effect to the other genes.It is the first time to compare the single-gene transformation and dual-gene transformation for their merits and faults. The regulation of multi-gene transformation will help us understand the mechanisms that control plant AsA accumulation and select the best combination, thus providing the opportunity to enhance nutritional quality and stress tolerance of crop plants.更多还原