【作者】 李婷；

【导师】 李晚忱；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 p-胡萝卜素(C40H56)是类胡萝卜素之一,是一种橘黄色脂溶性化合物,在自然界中稳定存在的天然色素。在许多的天然食物中均含有p-胡萝卜素,例如绿色蔬菜、甘薯、胡萝卜、菠菜等。p-胡萝卜素是维生素A合成的前体,具有免疫、抗氧化、预防癌症和延缓癌症等非常重要的生理功能。人体自身不能合成类胡萝卜素,主要依赖于饮食中类胡萝卜素的供应。但是,在许多植物中类胡萝卜素含量较低,尤其是p-胡萝卜素。玉米作为世界和我国第一大粮食作物,类胡萝卜素的含量较低,增加p-胡萝卜素的含量可显著提升玉米的营养价值。在高等植物中,p-胡萝卜素是在质体中通过类异戊二烯途径合成的。其生物合成过程包括缩合、脱氢、环化、羟基化以及环氧化等一系列反应。目前,催化这些反应的关键酶基因陆续克隆鉴定,为通过DNA重组技术和遗传工程生产p-胡萝卜素开辟了道路。本研究克隆出p-胡萝卜素生物合成的关键酶基因：八氢番茄红素合成酶基因PSY和八氢番茄红素脱氢酶基因CrtI,通过重叠PCR的方法连接成融合基因,构建成单子叶植物表达载体,利用农杆菌介导法转化玉米自交系“18-599”,以期获得β-胡萝卜素大量积累的植株。1.植物表达载体构建。根据NCBI上公布的PSY和CrtI基因序列,设计引物,以玉米的cDNA为模板克隆出PSY基因序列,以噬夏孢欧文氏菌的DNA为模板克隆出CrtI基因序列,人工合成豌豆核酮糖小亚基转运肽tp基因序列,通过重叠PCR将三段基因连接成融合基因并引入限制性酶切位点,定向插入到植物表达载体pTF101.1中,构建成单子叶植物表达载体。2.玉米愈伤组织培育及转化以玉米自交系“18-599”的幼胚为外植体,诱导培养胚性愈伤组织,通过农杆菌介导的方法转化,经除草剂抗性培养基梯度筛选后分化出T0代再生植株150株。3.再生植株的分子检测设计特异PCR引物,扩增目的基因1315bp片段,对抗性愈伤组织分化得到的150株T0代再生植株进行分子检测,有10个T0代植株表现阳性。本研究PCR初步检测结果,对阳性植株进一步筛选鉴定,可望培育富含β-胡萝卜素的玉米新种质。更多还原

【Abstract】 P-carotene (C40H56) is one of the carotenoids, which is an orange fat-soluble compounds. It is a stable natural pigment in the nature. Many natural foods contain β-carotene, such as:green vegetables, sweet potatoes, carrots, spinach. β-carotene plays an important role in physiological functions:it is the precursor of vitamin A, has the role of the immune and antioxidant, can prevent cancer and delay the development of cancer and so on.But the human body can not synthesize carotenoids, mainly dependent on diet. The content of carotenoid is relatively low in many plants, especially the β-carotene. Corn as the largest food in the world and China, corn endosperm has relatively low levels of carotenoids. Improve the amount of β-carotene can significantly improve the nutritional value of corn.In higher plants, β-carotene is synthesized by the isoprenoid pathway in plastids. The biosynthesis process includes condensation, dehydrogenation, cyclization, hydroxy, and epoxy, etc. At present, the key enzymes’genes of the catalytic reactions have been cloned and identified successively; it opened the way to product P-carotene by recombinant DNA technology and genetic engineering. This study cloned the key enzymes’ genes of P-carotene biosynthetic pathway:the gene of phytoene synthase (PSY) and phytoene dehydrogenase (CrtI), the two genes was connected fusion gene by the overlapping PCR method, the fusion gene was inserted into the monocot plant expression vector, the plant expression vector was shifted to the maize of "18-599" by Agrobacterium-mediated transformation, in order to get the plants with massive accumulation of P-carotene.1. The plant expression vector was constructedAccording to the PST and CrtI gene sequences which published on NCBI, the primers were designed. The PSY gene was cloned from the corn cDNA, the CrtI gene was cloned from the Erwinia uredovora DNA, the pea ribulose small subunit transit peptide gene sequences were syntheticed.We connected the three genes into a fusion gene by the overlapping PCR, at the same time introducted the restriction sites, Finally the fusion gene was inserted into the monocot plant expression vector pTF101.1.2. Cultivation and transformation of maize embryonic calli The explants is the immature embryo of "18-599", then was induced embryogenic callus,the callus was transformed by Agrobacterium-mediated.Finally got150To generation regenerated plants after herbicide resistance medium gradient screening.3. Molecular detection of regenerated plantsDesign specific PCR primers to amplify the target gene1315bp fragment,The To generation150regenerated plants were detected; there were10plants which were certified to be positive by PCR detection of target genes.Based on the results of PCR, the positive plants were identified for further; we expect to provide material for the new breed which is rich of P-carotene.更多还原