大豆尿黑酸叶绿醇转移酶（HPT）基因的分离与功能分析
摘要
维生素E又称生育酚,是仅由光合生物合成的一类脂溶性的强抗氧化剂,为人体所必需的维生素之一,它可以清除生物体内的活性氧和自由基。因此,食用维生素E可以增强机体免疫功能,具有防衰老、防癌、减缓多种恶性疾病(白内障、神经紊乱、心血管疾病等)及润肤美容的作用。但是,在植物当中生育酚的含量又普遍较低,因此,采用基因工程技术手段提高植物当中的生育酚的含量成为目前营养基因组学研究的热点。
大豆尿黑酸叶绿醇转移酶处于维生素E生物合成途径的关键一步,它的作用是催化叶绿醇焦磷酸(phytyl-PP)与尿黑酸(HGA)缩合生成生育酚的前体。随着维生素E代谢途径的阐明以及相关基因的克隆,人们开始通过分子生物学技术研究如何提高植物中α-生育酚的含量。为探索利用基因工程手段提高总生育酚含量,本研究采用RT-PCR技术,从大豆中分离了尿黑酸叶绿醇转移酶的全长cDNA序列,该cDNA序列全长1200bp。为了进一步研究该基因的功能,我们得到了以下结果:
1.提取大豆总RNA,利用RT-PCR技术,从大豆中分离了大豆尿黑酸叶绿醇转移酶(HPT)的全长cDNA序列。
2.构建了大豆基因的植物表达载体pBI121-HPT,通过真空渗透法转化拟南芥,获得转基因植株35株,PCR检测阳性率是51.2%。通过自交和抗性筛选标记获得了转基因纯系植株。
3.利用HPLC测定转基因植株的总生育酚含量,结果显示,转基因植株的总生育酚含量普遍高于野生型植株,提高3-6倍。
Vitamin E, commonly known as Tocopherols, is a class of lipid-soluble antioxidants synthesized only by plants and other photosynthetic organisms. Tocopherols are essential components of the human diet because they perform numerous critical functions including quenching and scavenging various reactive oxygen species and free radicals. Because of these and other activities, dietary tocopherols are thought to play an important role in improving immune function and in limiting the incidence and progression of several degenerative human diseases including certain types of cancer, cataracts, neurological disorders, and cardiovascular disease. The yield of tocopherol in the plant is commonly low, so enhancing the yield of tocopherol in the plant with gene engineering means will become a research hotspot in the field of nutrition genomics.
Homogengtisate phytyltranferase (HPT), one of the key enzymes in tocopherol biosynthetic pathway in plants, catalyzing condensation of PDP and HGA in the first set of tocopherol biosynthesis.With understanding biosynthetic pathway of vitamin E and being isolated correlated gene, it is focus on how to increaseα-tocopherols level by molecular biotechnology. To explore how to enhanceα-tocopherols level and identify the function of homogengtisate phytyltranferase in Soybean, the full-length cDNA of HPT, 1200bp length was obtained from Sobean by RT-PCR. The present research employs a transgenic approach to further analyze the function of HPT.
The results obtained through the present research are as follows:
1. The total RNA was extracted from Soybean and the full-length cDNA of HPT gene was isolated by RT-PCR
2. A recombinant vector pBI121-HPT was constructed for HPT gene expression in plant and was transformed into the genome of Arabidopsis via agro-bacterium mediated transformation by vacuum infiltration. 35 kanamycin-resistant plants were obtained and 51.2% of them are PCR positive. Homozygous HPT gene transgenic line were obtained and were used as the material in the later analysis.
3. Theα-tocopherols was analysised by HPLC, in leaves, HPT overexpression resulted in a 3-6 fold increase relative to wild type.
大豆尿黑酸叶绿醇转移酶处于维生素E生物合成途径的关键一步,它的作用是催化叶绿醇焦磷酸(phytyl-PP)与尿黑酸(HGA)缩合生成生育酚的前体。随着维生素E代谢途径的阐明以及相关基因的克隆,人们开始通过分子生物学技术研究如何提高植物中α-生育酚的含量。为探索利用基因工程手段提高总生育酚含量,本研究采用RT-PCR技术,从大豆中分离了尿黑酸叶绿醇转移酶的全长cDNA序列,该cDNA序列全长1200bp。为了进一步研究该基因的功能,我们得到了以下结果:
1.提取大豆总RNA,利用RT-PCR技术,从大豆中分离了大豆尿黑酸叶绿醇转移酶(HPT)的全长cDNA序列。
2.构建了大豆基因的植物表达载体pBI121-HPT,通过真空渗透法转化拟南芥,获得转基因植株35株,PCR检测阳性率是51.2%。通过自交和抗性筛选标记获得了转基因纯系植株。
3.利用HPLC测定转基因植株的总生育酚含量,结果显示,转基因植株的总生育酚含量普遍高于野生型植株,提高3-6倍。
Vitamin E, commonly known as Tocopherols, is a class of lipid-soluble antioxidants synthesized only by plants and other photosynthetic organisms. Tocopherols are essential components of the human diet because they perform numerous critical functions including quenching and scavenging various reactive oxygen species and free radicals. Because of these and other activities, dietary tocopherols are thought to play an important role in improving immune function and in limiting the incidence and progression of several degenerative human diseases including certain types of cancer, cataracts, neurological disorders, and cardiovascular disease. The yield of tocopherol in the plant is commonly low, so enhancing the yield of tocopherol in the plant with gene engineering means will become a research hotspot in the field of nutrition genomics.
Homogengtisate phytyltranferase (HPT), one of the key enzymes in tocopherol biosynthetic pathway in plants, catalyzing condensation of PDP and HGA in the first set of tocopherol biosynthesis.With understanding biosynthetic pathway of vitamin E and being isolated correlated gene, it is focus on how to increaseα-tocopherols level by molecular biotechnology. To explore how to enhanceα-tocopherols level and identify the function of homogengtisate phytyltranferase in Soybean, the full-length cDNA of HPT, 1200bp length was obtained from Sobean by RT-PCR. The present research employs a transgenic approach to further analyze the function of HPT.
The results obtained through the present research are as follows:
1. The total RNA was extracted from Soybean and the full-length cDNA of HPT gene was isolated by RT-PCR
2. A recombinant vector pBI121-HPT was constructed for HPT gene expression in plant and was transformed into the genome of Arabidopsis via agro-bacterium mediated transformation by vacuum infiltration. 35 kanamycin-resistant plants were obtained and 51.2% of them are PCR positive. Homozygous HPT gene transgenic line were obtained and were used as the material in the later analysis.
3. Theα-tocopherols was analysised by HPLC, in leaves, HPT overexpression resulted in a 3-6 fold increase relative to wild type.
引文
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2 Fukuzawa K, Gebicky JM ,Oxidation of _-tocopherol in micelles and liposomes by the hydroxyl, perhydroxyl, and superoxide free radicals. Arch Biochem Biophys ,1983,226: 242–251
3 Fryer, M.J. The antioxidant effects of thylakoid vitamin E(a-tocopherol). Plant Cell Environ. 1992,15, 381–392.
4 Traber, M.G. and Sies, H. Vitamin E in humans: demand and delivery. Annu. Rev. Nutr. 1996,16, 321–347.
5 Pryor WA .Vitamin E and heart disease: basic science to clinical intervention trials. Free Radic Biol Med.2000,28: 141–164
6 Traber, M.G. and Sies, H. Vitamin E in humans: demand and delivery. Annu. Rev. Nutr. 1996, 16, 321-347.
7 BramLey PM, Elmadfa I, Kafatos A, Kelly FJ, Manios Y, Roxborough HE,Schuch W, Sheehy PJA, Wagner KH .Vitamin E. J Sci Food Agric.2000,80: 913–938 .
8 Yamauchi, R., and Matsushita, S. Light induced lipid peroxidation in isolated chloroplasts from spinach leaves and role of alpha-tocopherol vitamin E. Agric. Biol. Chem.1979, 43:2157-2161.
9 Fryer MJ.Evidence for the photoprotective effects of vitamin E. Photochem Photobiol .1993,58: 304–31
10尤新.天然维生素E的生理功能和开发前景分析.中国食品添加剂,2000,3:30-32
11 Pongracz G, Weiser H, Matzinger D,Tocopherols—natural antioxidants. Fett Wiss Technol ,1995,97:90–104
12 Sheppard AJ, Pennington JA, Weihrauch JL ,Analysis and distribution of Vitamin E in vegetable oils and foods. In: Packer L, Fuchs J (eds) Vitamin E in health and disease. Marcel Dekker, New York, pp 1993,9–31
13 Tyamagondlu V., Identification and characterization of an Arabidopsis homogentisate phytyltransferase paralog, planta,2006, 223: 1134–1144
14 Grusak MA, DellaPenna D ,Improving the nutrient composition of plants to enhance human nutritionand health. Annu Rev Plant Physiol Plant Mol Biol,1999, 50: 133–161
15 Okada K, Saito T, Nakagawa T, et al. Five geranylgeranyl diphosphate synthase expressed in different organs are localized into three subcellular compatments in Arabidopsis. Plant Physiol, 2000,122(4):1045-1056.
16 Kamal2Eldin A , Appelqvist LA. The chemistry and antioxidantproperties of tocopherols and tocotrienols. L ipi ds , 1996 ,31 : 671～701
17 Soll J, Douce R, Schultz G. Joyard J, Localization and synthesis of Prenylquinones in isolated outer and inner envelope membranes from spinach chloroplasts. Arch Biochen Biophys,1985,238(1):290-299
18 Soll J, Douce R, Schulta G.Site of Biosynthesis ofα-tocopherol in spinach chloroplasts. FEBS Lett, 1980,112(2):243-246
19 National Research Council ( U. S. ) , Food and Nutrition Board. NAS2NRC Recommended Dietary Allowances. 10th ed. Washington , DC: National Academy Press , 1989. 284
20 Peter Dormann. Functional diversity of tocochromanols in plants. Planta. 2007,225.269-276
21 Tangney CC. Vitamin E and cardiovascular disease. Nutr Today ,1997 ,32 :13～22
22 Lichtenthaler, H.K., Prenzel, U., Douce, R. and Joyard, J. (1981)Localization of prenylquinones in the envelope of spinach chloroplasts. Biochim. Biophys. Acta 641, 99–105.
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