小麦ζ-胡萝卜素脱饱和酶（ZDS）基因的克隆及序列分析
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摘要
类胡萝卜素(carotenoids)是自然界存在的一类由异戊二烯为结构单元组成的化合物的一大类家族的统称。该类化合物广泛分布于部分细菌、真菌、植物等生物体内。类胡萝卜素主要分为胡萝卜素及其氧化物叶黄素两大类。很多类胡萝卜素跟人类的健康有重要的关系,特别是β-胡萝卜素以及番茄红素对眼睛的发育、抵御癌症、提高免疫力均有很重要的作用。绝大多数类胡萝卜素是C40类的碳氢四萜类(胡萝卜素)及其含氧衍生物(叶黄素),它们通过类异戊二烯途径中的部分途径合成。在高等植物中类胡萝卜素的合成途径分为两条:一条称MVA途径,另一条称MEP途径。ζ-胡萝卜素去饱和酶(ZDS)是类胡萝卜素生物合成途径上游控制β-胡萝卜素生成的关键酶,它催化9,9'-双顺-ζ-胡萝卜素脱氢生成7,9,7',9'-四顺式-番茄红素。
本研究以小麦(Triticum aestivum)叶片为材料,应用RT-PCR及RACE的方法成功克隆了小麦ZDS蛋白基因的完整阅读框序列。该开放阅读框(ORF)由1,707个碱基组成,编码568个氨基酸残基组成的蛋白质,理论分子量约为62.5 kDa,等电点为6.80,N-端有一个包含一个典型的氨基氧化酶的结构域;二级结构预测表明,不规则盘绕和α-螺旋是ZDS蛋白中主要的结构元件;同源序列比对表明其与多种植物zds基因具有较高的序列同源性,其中水稻(Oryza sativa)、玉米(Zea mays)、拟南芥(Arabidopsis thaliana)、胡萝卜(Daucus carota)同源性分别为92%、93%、83%、88%。。证实该基因确为zds基因。进化树分析显示小麦ZDS与玉米和水稻ZDS有很高的亲缘关系。
该研究结果为进一步研究该基因的表达、功能和通过基因工程手段提高小麦营养价值打下了良好的基础。
Carotenois are a large group of natural products that are most formally built from isoprenic unit. Those kinds of compounds are biosynthesized by some bacterium, fungi, and plants. Carotenois mainly consist of two large groups: carotenes and its oxidative derivatives(xanthophylls). Many of these carotenois are closely related to human health, especially theβ-carotene and lycopene are very important to the development of eyes, prohibiting cancers and elevating immunity. They are synthesized through part of the isoprenoids pathway. In higher plant their biosynthesis pathway includes two :one is MVA pathway,the other is MEP pathway.ζ-catotene desaturase(ZDS) is a importanr enzyme that controls the biosynthesis ofβ-carotene upstream. It catalyses the conversion of 9,9,-di-ζ-catotene to 7,9,7,9,-tetra-lycopene.
In this work, reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques had been applied to clone the total open reading frame of the zds gene using wheat (Triticum aestivum) leaves as the target material. The full-length of open reading frame is 1,707 bp and encodes a peptide containing 568 amino acid residues, with a theoretical molecular weight of 62.5 kDa, an isoelectric point of 6.80 and an amino oxidase domain at its N-terminal. Secondary structure prediction reveals thatα-helix and random coil are the main structural conformations in ZDS protein. Through sequence analysis by BlastN online, the wheat zds nucleiotide sequence showed highly homologous to the genes which are from other plant species. The homology that compared with Oryza sativa, Zea mays, Arabidopsis thaliana, Daucus carota were 92%, 93%, 83% and 88% respectively. The phylogenetic tree showed that wheat ZDS was closely related to rice ZDS and maize ZDS.
The cloning and analysis of wheat zds gene has layed a good basis for research on functional expression and potential application in improving the nutrition value of wheat through genetic engeering.
本研究以小麦(Triticum aestivum)叶片为材料,应用RT-PCR及RACE的方法成功克隆了小麦ZDS蛋白基因的完整阅读框序列。该开放阅读框(ORF)由1,707个碱基组成,编码568个氨基酸残基组成的蛋白质,理论分子量约为62.5 kDa,等电点为6.80,N-端有一个包含一个典型的氨基氧化酶的结构域;二级结构预测表明,不规则盘绕和α-螺旋是ZDS蛋白中主要的结构元件;同源序列比对表明其与多种植物zds基因具有较高的序列同源性,其中水稻(Oryza sativa)、玉米(Zea mays)、拟南芥(Arabidopsis thaliana)、胡萝卜(Daucus carota)同源性分别为92%、93%、83%、88%。。证实该基因确为zds基因。进化树分析显示小麦ZDS与玉米和水稻ZDS有很高的亲缘关系。
该研究结果为进一步研究该基因的表达、功能和通过基因工程手段提高小麦营养价值打下了良好的基础。
Carotenois are a large group of natural products that are most formally built from isoprenic unit. Those kinds of compounds are biosynthesized by some bacterium, fungi, and plants. Carotenois mainly consist of two large groups: carotenes and its oxidative derivatives(xanthophylls). Many of these carotenois are closely related to human health, especially theβ-carotene and lycopene are very important to the development of eyes, prohibiting cancers and elevating immunity. They are synthesized through part of the isoprenoids pathway. In higher plant their biosynthesis pathway includes two :one is MVA pathway,the other is MEP pathway.ζ-catotene desaturase(ZDS) is a importanr enzyme that controls the biosynthesis ofβ-carotene upstream. It catalyses the conversion of 9,9,-di-ζ-catotene to 7,9,7,9,-tetra-lycopene.
In this work, reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques had been applied to clone the total open reading frame of the zds gene using wheat (Triticum aestivum) leaves as the target material. The full-length of open reading frame is 1,707 bp and encodes a peptide containing 568 amino acid residues, with a theoretical molecular weight of 62.5 kDa, an isoelectric point of 6.80 and an amino oxidase domain at its N-terminal. Secondary structure prediction reveals thatα-helix and random coil are the main structural conformations in ZDS protein. Through sequence analysis by BlastN online, the wheat zds nucleiotide sequence showed highly homologous to the genes which are from other plant species. The homology that compared with Oryza sativa, Zea mays, Arabidopsis thaliana, Daucus carota were 92%, 93%, 83% and 88% respectively. The phylogenetic tree showed that wheat ZDS was closely related to rice ZDS and maize ZDS.
The cloning and analysis of wheat zds gene has layed a good basis for research on functional expression and potential application in improving the nutrition value of wheat through genetic engeering.
引文
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[5]惠伯棣,朱雨杰,武兴德,陈杭.高等植物中类胡萝卜素的生物合成.北京农业科学, 1999, 17(2):17-20.
[6] Howitt CA, Pogson BJ. Carotenoid accumulation and function in seeds and non-green tissues. Plant Cell and Environment, 2006, 29:435-445.
[7]郑阳霞,杨婉身,季静,王罡.植物类胡萝卜素生物合成及其相关基因在基因工程中的应用.细胞生物学杂志, 2006, 28(3):442-446.
[8] Bartley GE, Scolnik PA. Molecular biology of carotenoid biosynthesis in plants. Annual Review of Plant Physiology and Plant Molecular Biology, 1994, 45:287-301.
[9] Tracewell CA, Vrettos JS, Bautista JA, Frank HA, Brudvig GW. Carotenoid photooxidation in photosystem II. Archives of Biochemistry and Biophysics, 2001, 385 (1):61-69.
[10] Olson JA, Krinsky NI. The colorful, fascinating world of the carotenoids: important physiologic modulators. Federation of American Societies for Experimental Biology, 1999, 9 (15): 1547-1550.
[11] Demmig-Adams B, Gilmore AM, Adams WW III. In vivo functions of carotenoids in higher plants. Federation of American Societies for Experimental Biology, 1996,10(4):403-412.
[12]许青等.类胡萝卜素研究进展.国外医学生理、病理科学与临床分册, 1996, 16(4):249-251.
[13]黄彬城,季静,王罡,郑阳霞.植物类胡萝卜素的研究进展.天津农业科学, 2006, 12(2):13-17.
[14] Van den Berg H et al. The potential for the improvement of carotenoid levels in foods and the likely systemic effects. Journal of the Science of Food and Agriculture, 2000, 80:880-912.
[15] Bendich A, Olson JA. Biological action of carotenoids. Federation of American Societies for Experimental Biology, 1989, 3(8):1927-1932.
[16] Lee C, McCoon P, LeBowitz J.. Vitamin A value of sweet corn. Journal of Agriculture and Food Chemistry, 1981, 29 (6):1294-1295.
[17] Fawzi WW, Hunter DJ: Vitamins in HIV Disease Progression and Vertical Transmission, Epidemiology, 1998, 9(4):457-466.
[18] Ye X, Al-babili S, Kloti A et al. Engineering provitamin(β-carotene)biosynthetic pathway into rice endosperm. Science, 2000, 287(14):303-305.
[19] Agarwal S, Rao AV. Tomato lycopene and low density lipoprotein oxidation:a human dietary intervention study.Lipids, 1998, 33(10):981-984.
[20] Gann PH, Khachik F. Tomatoes or lycopene versus prostate cancer:is evolution anti -reductionist[J]. Journal of the National Cancer Institute, 2003, 95:1563-1565.
[21] Hadley CW, Clinton SK, Schwartz SJ. The consumption of processed tomato products enhances plasma lycopene concentrations in association with a reduced lipoprotein sensitivity to oxidative damage. Journal of Nutrition, 2003, 133(3):727-732.
[22] Cheryl L Rock. Caroteniods:biology and treatment.Pharmacology and Therapeutics, 1997, 75 (3):185-197.
[23]韩雅珊.类胡萝卜素的功能研究进展.中国农业大学学报, 1999, 4(1):5-9.
[24] McDermott JH. Antioxidant nutrients:current dietary recommendations and research update. Journal of the American Pharmaceutical Association, 2000, 40(6):785-799.
[25]田允波.维生素A胡萝卜素与牛的繁殖机理.甘肃畜牧兽医, 1991(2):28-30.
[26] Tjoelker LW, Chew BP, Tanaka TS and Daniel LR. Bovine vitamin A and ?-carotene intake and lactational status:Responsiveness of peripheral blood polymorphonuclear leukocytes to vitamin A andβ-carotene challenge in dairy cows during the early dry period. Journal of Diary Science, 1988, 71:3112-3119.
[27] Soper FF, Muscoplat CC and Johnson DW. In vitro stimulation of bovine peripheral blood lymphocytes: analysis of variation of lymphocyte blastogenic response in normal dairy cattle. American Journal of Veterinary Research, 1978,39(6):1039-1042.
[28] Gerster H. Potential role of beta-cartotene in the prevention of cardiovascular disease. Annals of the New York Academy of Sciences, 1993, 691:10-19.
[29]陈炳辉.类胡萝卜素的特性与应用.科学发展月刊, 1989(6):599-603.
[30] Clinton SK, Giovannucci E. Diet, nutrition and prostate cancer. Annual Review of Nutrition, 1998, 18:413-440.
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