奇异果甜蛋白thaumatin基因在玉米中的表达以及thaumatin-PDI二价载体在烟草中表达的研究
摘要
奇异果甜蛋白Thaumatin是从一种叫Thaumatococcus daniellii Benth的竹芋科(Marabtaceae)植物的果实中提取出来的。它具有无毒安全、甜度高、热量低等许多优点，是一种新型甜味剂。但由于Thaumatococcus daniellii Benth对生长条件极其苛刻，在原产地之外的地方无法结实，试图通过人工栽培Thaumatococcus daniellii Benth大量提取Thaumatin是不可能的，于是人们便把希望寄托在thaumatin的基因工程上。三十年来，人们把thaumatin基因转入不同的宿主，包括大肠杆菌、真菌和高等植物等，希望获得大量重组的具有活性的thaumatin，但结果并不理想，重组蛋白不是没甜昧就是产量很低，尤其在高等植物中难度更大。
本文通过基因工程技术，构建了植物表达载体pCAMBIA1302-tha，测序证明其正确性。将pCAMBIA1302-tha转入农杆菌LBA4404，侵染玉米18红的胚性愈伤组织，通过组培技术得到玉米阳性植株。
提取玉米18红阳性植株基因组DNA，分别用PCR、PCR-Southern和Southern杂交检测转基因植株，结果显示thaumatin cDNA基因已整合到转基因玉米中。用转基因玉米的总RNA通过RT-PCR反转录扩增出thaumatin cDNA基因，其Northern blotting检测出现目的杂交条带，证明在35S启动子及其它调控元件的控制下，thaumatin cDNA基因在转基因玉米中转录出mRNA，转基因玉米可溶性总蛋白的SDS-PAGE检测也发现相应分子量的蛋白条带。最后甜味品尝发现转基因玉米尚无甜味，证明thaumatin cDNA基因在转基因玉米中还未表达成有活性的甜味蛋白。
显然，外源甜味蛋白基因thaumatin在玉米中未能表达成有活性的蛋白与很多原因有关，例如，Thaumatin蛋白来源于Thaumalococcus daniellii Benth果实的假种皮中，所以预期表达部位可能是玉米穗部，实验还未及对穗部进行甜味检测，所以还不能定论所得转基因玉米不甜。总之，通过本实验独创了一套切实可行的农杆菌介导转化玉米胚性愈伤组织的可靠方法。
Thaumatin, 'the sweetest substance known', is a protein produced by the plant Thaumatococcus daniellii Benth. It is an intensely sweet protein (100,000 times sweeter than sucrose on a molar basis), which has been approved for human consumption, and is currently used in products such as chewing gum, dairy produce and Pharmaceuticals because of its low calories, high sweetness and innocuity. However, Thaumatococcus daniellii Benth can't seed outside its producing area. But gene engineering can solve this problem.
Maize is a main crop. If sweet enough, the maize can promote the livestock eating and weighting. So I wish, through the gene engineering technique, the thaumatin can express in the maize. Meanwhile, the system, the high efficiency transformation of maize mediated by Agrobacterium tumefaciens, will be further improved.
After successfully constructing a new botanic expressing vector pCAMBIA1302-tha containing the sweet-protein thaumatin cDNA gene, the recombinant plasmid was transformed into the calli of maize R18 via agrobacterium LBA4404. Through the organ culture technology, we have gained the regenerating transgenic plants. The genome DNA of transformed maize's leaves was extracted. It proved that thaumatin cDNA gene has integrated into the genome of transformed maize plants by PCR, PCR-Southern and Southern blotting analysis. Extracting the whole RNA from the leaves of transgenic plants, and the RT-PCR and Northern blotting results confirmed that the thaumatin cDNA gene was transcripted into mRNA. Isolating the all dissolvable proteins from the leaves of these plants. The result of SDS-PAGE showed the same molecular weight protein as Thaumatin in the transgenic maize, not in untransgenic maize. On the whole, it illuminates the thaumatin cDNA gene has translated into the protein, only we can't confirm it is Thaumatin. At present, because we haven't gained the antibody of Thaumatin, it cannot be proved that it is Thaumatin by the way of Western blotting analysis. At the same time, the leaves of these transgenic maize were not sweet. All of the words, the thaumatin cDNA gene has not expressed the active protein in the leaf cells of transgenic maize.
We have not gained the sweet maize. Of course, it is due to a lot of reasons. For example, the fringes of transgenic maize (The Thaumatin is produced by the plant Thaumatococcus daniellii Benth in the gelatinous arils of the fruit, so we expect it will correctly express in the fringes of transgenic maize), which have not maturated, have not done the sweet analysis. So it cannot confirm the transgenic maize unsweet. But we form the transformation system of maize mediated by Agrobacterium tumefaciens.
本文通过基因工程技术，构建了植物表达载体pCAMBIA1302-tha，测序证明其正确性。将pCAMBIA1302-tha转入农杆菌LBA4404，侵染玉米18红的胚性愈伤组织，通过组培技术得到玉米阳性植株。
提取玉米18红阳性植株基因组DNA，分别用PCR、PCR-Southern和Southern杂交检测转基因植株，结果显示thaumatin cDNA基因已整合到转基因玉米中。用转基因玉米的总RNA通过RT-PCR反转录扩增出thaumatin cDNA基因，其Northern blotting检测出现目的杂交条带，证明在35S启动子及其它调控元件的控制下，thaumatin cDNA基因在转基因玉米中转录出mRNA，转基因玉米可溶性总蛋白的SDS-PAGE检测也发现相应分子量的蛋白条带。最后甜味品尝发现转基因玉米尚无甜味，证明thaumatin cDNA基因在转基因玉米中还未表达成有活性的甜味蛋白。
显然，外源甜味蛋白基因thaumatin在玉米中未能表达成有活性的蛋白与很多原因有关，例如，Thaumatin蛋白来源于Thaumalococcus daniellii Benth果实的假种皮中，所以预期表达部位可能是玉米穗部，实验还未及对穗部进行甜味检测，所以还不能定论所得转基因玉米不甜。总之，通过本实验独创了一套切实可行的农杆菌介导转化玉米胚性愈伤组织的可靠方法。
Thaumatin, 'the sweetest substance known', is a protein produced by the plant Thaumatococcus daniellii Benth. It is an intensely sweet protein (100,000 times sweeter than sucrose on a molar basis), which has been approved for human consumption, and is currently used in products such as chewing gum, dairy produce and Pharmaceuticals because of its low calories, high sweetness and innocuity. However, Thaumatococcus daniellii Benth can't seed outside its producing area. But gene engineering can solve this problem.
Maize is a main crop. If sweet enough, the maize can promote the livestock eating and weighting. So I wish, through the gene engineering technique, the thaumatin can express in the maize. Meanwhile, the system, the high efficiency transformation of maize mediated by Agrobacterium tumefaciens, will be further improved.
After successfully constructing a new botanic expressing vector pCAMBIA1302-tha containing the sweet-protein thaumatin cDNA gene, the recombinant plasmid was transformed into the calli of maize R18 via agrobacterium LBA4404. Through the organ culture technology, we have gained the regenerating transgenic plants. The genome DNA of transformed maize's leaves was extracted. It proved that thaumatin cDNA gene has integrated into the genome of transformed maize plants by PCR, PCR-Southern and Southern blotting analysis. Extracting the whole RNA from the leaves of transgenic plants, and the RT-PCR and Northern blotting results confirmed that the thaumatin cDNA gene was transcripted into mRNA. Isolating the all dissolvable proteins from the leaves of these plants. The result of SDS-PAGE showed the same molecular weight protein as Thaumatin in the transgenic maize, not in untransgenic maize. On the whole, it illuminates the thaumatin cDNA gene has translated into the protein, only we can't confirm it is Thaumatin. At present, because we haven't gained the antibody of Thaumatin, it cannot be proved that it is Thaumatin by the way of Western blotting analysis. At the same time, the leaves of these transgenic maize were not sweet. All of the words, the thaumatin cDNA gene has not expressed the active protein in the leaf cells of transgenic maize.
We have not gained the sweet maize. Of course, it is due to a lot of reasons. For example, the fringes of transgenic maize (The Thaumatin is produced by the plant Thaumatococcus daniellii Benth in the gelatinous arils of the fruit, so we expect it will correctly express in the fringes of transgenic maize), which have not maturated, have not done the sweet analysis. So it cannot confirm the transgenic maize unsweet. But we form the transformation system of maize mediated by Agrobacterium tumefaciens.
引文
F．奥斯伯，R．布伦特，R．E．金斯顿等著，颜子颖，王海林译．精编分子生物学实验指南，科学出版社，2001
范长胜，陈永春，李爽等．超甜蛋白的基因工程及开发研究进展，工业微生物，1999，29(1)：29～33
郭尧君，蛋白质电泳实验技术，科学出版社，1999
J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯著，金冬雁，黎孟枫等译．分子克隆实验指南(第二版)，科学出版社，2002
皮灿辉等．提高转基因植物外源基因表达效率的途径，中国生物工程杂志，2003，23(1)：1～5
卢圣栋．现代分子生物学实验技术，高等教育出版社，1993
施建科，叶蕴华，田桂玲．有甜味的蛋白质[J]，化学通报，1998，8：21～25
王关林，方宏筠．植物基因工程原理与技术，科学出版社，1998
王家政，范明．蛋白质技术手册，科学出版社，2000
吴乃虎，基因工程原理(第二版)，科学出版社，2001
杨成丽，刘德立等．植物甜蛋白thaumatin研究进展，武汉植物学研究，2001，19(2)：153～157
杨美珠，潘乃穟，陈章良．高效马铃薯遗传转化体系的建立及甜蛋白基因的导入，植物学报，1992，34(1)：31～36
杨奇志，赵琦等．基因枪技术在农作物基因转化中的应用和进展，生物技术通报，2003，6：4～17
印莉萍，刘祥林．分子细胞生物学实验技术，首都师范大学出版社，2001
张初，吴光耀．甜蛋白与抗虫蛋白，生物学通报，1996，31(9)：8～11
周平，潘乃穟等．外源甜蛋白Thaumatin基因转入烟草的研究，植物学通报，1994，11(4)：17～20
Daniell S, Mellits K.H, et al; Refolding the sweet-tasting protein thaumatin Ⅱ from insoluble inclusion bodies synthesized in Escherichia coli, Food Chemistry, 2000, 71: 105～110.
Dolgov S V, Lebedev V G, Firsov A P, et al. Expression of thaumatin Ⅱ gene in horticultural crops.Genetics and breeding for crop quality and resistance. Proceedings of the X V EUCARPIA Congress,Viterbo, Italy, September 20-25, 1998, 1999:165～172.
Edens L, Born I, et al. Synthesis and processing of the plant protein thaumatin in yeast, Cell, 1984,37:629～633.
Edens L, Heslinga L, Klok R, et al; Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli,Gene, 1982,18:1～12.
Edens L, van der Wel H. Microbial synthesis of the sweet-tasting plant protein thaumatin, Trends in Biotechnology,1985,3:61～63.
Etheridge K. The sales and marketing of talin.In"Thaumatin", eds, Witty M.and Higginbotham J D, Boca Raton, Florida. CRC Press, 1994, pp.47～59.
Faus I.Recent developments in the characterization and biotechnological production of sweet-tasting proteins, Applied Microbiology and Biotechnology, 2000, 53(2): 145～151.
Faus I, del Moral C, et al; Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var.awamori, Appl Microbiol Biotechnol, 1998, 49:393～398.
Faus I, Patio C, Del Ro J L, et al; Expression of a synthetic gene encoding the sweet-tasting protein thaumatin in the filamentous fungus Penicillium roquefortii, Biotechnology Letters, 1997,19:1185～1191.
Hahm Y T, Batt C A. Expression and Secretion of thaumatin in Aspergillus oryzae, Agric Biol Chem, 1990, 54(10):2513～2520.
Higginbotham J D. Hough C A M. In Sensory properties of foods (Birch G C, Brennan J G, Parker K J,Eds), London. Elsevier Applied Science Press, 1977,pp. 129～149.
Illingworth C, Larson G, Hellekant G. Secretion of the sweet-tasting plant protein thaumatin by Bacillus subtilis,Biotechnol. Lett. 1988, 10:587～592.
Illingworth C, Larson G, Hellekant G. Secretion of the sweet-tasting plant protein thaumatin by Streptomyces lividans,J.,Ind, Microbiol. 1989, 4:37～42.
Kaneko R, Kitabatake N, Sweetness of sweet-protein thaumatin is more thermoresistant under acid conditions than under neutral or alkaline conditions, Biosci Biotechnol Biochem, 2001, 65(2): 409～413.
Kaneko R, Kitabatake N. Structure-sweetness relationship in thaumatin: importance of lysine residues, Chem Senses, 2001, 26:167～177.
Lee J H, Weiekmann J L, Koduri R K, et al; Expression of synthetic thaumatin genes in yeast,Biochemistry, 1988,27:5101～5107.
Lindemann B. Receptors and transduction in taste, Nature, 2001, 413:219～225.
Lyengar K B, Smits P, van der Ouderaa F J, et al; The complete amino acid sequence of the sweet-tasting protein thaumatin I, Eur J Biochem., 1979,96:193～204.
Malepszy S, Szwaeka M, Hrazdina G. Evaluation of transgenic cucumbers expressing the thaumatin gene, Use of agriculturally important genes in biotechnology, Proceeding of the NATO Advanced Research Workshop,Szeged,Hungary, 17-21 October 1999, 2000:131～134.
Margolskee R F. Molecular mechanisms of bitter and sweet taste transduction. J Bio Chem, 2002,277(1): 1～4.
Mitsuhara I, Ugaki M, et al. Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledomous plants. Plant Cell Physiol, 1996,37(1): 49～59.
Moralejo F J, Cardoza R E, Gutierrez S, et al;Thaumatin production in Aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage,Applied and Environmental Microbiology, 1999,65(3): 1168～1174.
Moralejo F J, Cardoza R E, Gutierrez S, et al; Overexpression and lack of degradation of thaumatin in an aspergillopepsin A-defective mutant of Aspergillus awamori containing an insertion in the peA gene, Appl Microbiol Biotechnol, 2000, 54:772～777.
Moralejo F J, Watson A J, ,leenes D J, et al; A defined level of protein disulfide isomerase expression is required for optimal secretion of thaumatin by Aspergillus awamori, Mol Genet Genomics, 2001, 266:246～253.
Slootstra J W, de Geus P, et al; Possible activite site of the sweet-tasting protein thaumatin. Chem Senses,1995,20:535～543.
Suami T, Hough L, Maehinami T, et al; Molecular mechanisms of sweet taste7:The sweet protein, Thaumatin Ⅰ, Food Chemistry, 1997, 60(3): 277～285.
van tier Wel H. Structure-activity relationships in the thaumatin molecule.In "Thaumatin"eds,M Witty, J D Higginbotham,Boca Raton. CRC Press, 1994,pp.115～122.
van der Wel H, Loeve K. Isolation and Characterization of thaumatin Ⅰ and Ⅱ, the sweet-tasting proteins from Thaumatococcus daniellii Benth. Eur J Biochem., 1972,31: 221～225.
Wallroth M, Gerats G M, Rogers S G, et al. Chromosomal localization of foreign genes in Petunia hybrida. Mol Gen Genet. 1986, 202: 146～152.
Witty M. Thaumatin Ⅱ—a palatability protein,Trends in Biotechnology, 1990, 8(5): 113～116.
范长胜，陈永春，李爽等．超甜蛋白的基因工程及开发研究进展，工业微生物，1999，29(1)：29～33
郭尧君，蛋白质电泳实验技术，科学出版社，1999
J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯著，金冬雁，黎孟枫等译．分子克隆实验指南(第二版)，科学出版社，2002
皮灿辉等．提高转基因植物外源基因表达效率的途径，中国生物工程杂志，2003，23(1)：1～5
卢圣栋．现代分子生物学实验技术，高等教育出版社，1993
施建科，叶蕴华，田桂玲．有甜味的蛋白质[J]，化学通报，1998，8：21～25
王关林，方宏筠．植物基因工程原理与技术，科学出版社，1998
王家政，范明．蛋白质技术手册，科学出版社，2000
吴乃虎，基因工程原理(第二版)，科学出版社，2001
杨成丽，刘德立等．植物甜蛋白thaumatin研究进展，武汉植物学研究，2001，19(2)：153～157
杨美珠，潘乃穟，陈章良．高效马铃薯遗传转化体系的建立及甜蛋白基因的导入，植物学报，1992，34(1)：31～36
杨奇志，赵琦等．基因枪技术在农作物基因转化中的应用和进展，生物技术通报，2003，6：4～17
印莉萍，刘祥林．分子细胞生物学实验技术，首都师范大学出版社，2001
张初，吴光耀．甜蛋白与抗虫蛋白，生物学通报，1996，31(9)：8～11
周平，潘乃穟等．外源甜蛋白Thaumatin基因转入烟草的研究，植物学通报，1994，11(4)：17～20
Daniell S, Mellits K.H, et al; Refolding the sweet-tasting protein thaumatin Ⅱ from insoluble inclusion bodies synthesized in Escherichia coli, Food Chemistry, 2000, 71: 105～110.
Dolgov S V, Lebedev V G, Firsov A P, et al. Expression of thaumatin Ⅱ gene in horticultural crops.Genetics and breeding for crop quality and resistance. Proceedings of the X V EUCARPIA Congress,Viterbo, Italy, September 20-25, 1998, 1999:165～172.
Edens L, Born I, et al. Synthesis and processing of the plant protein thaumatin in yeast, Cell, 1984,37:629～633.
Edens L, Heslinga L, Klok R, et al; Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli,Gene, 1982,18:1～12.
Edens L, van der Wel H. Microbial synthesis of the sweet-tasting plant protein thaumatin, Trends in Biotechnology,1985,3:61～63.
Etheridge K. The sales and marketing of talin.In"Thaumatin", eds, Witty M.and Higginbotham J D, Boca Raton, Florida. CRC Press, 1994, pp.47～59.
Faus I.Recent developments in the characterization and biotechnological production of sweet-tasting proteins, Applied Microbiology and Biotechnology, 2000, 53(2): 145～151.
Faus I, del Moral C, et al; Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var.awamori, Appl Microbiol Biotechnol, 1998, 49:393～398.
Faus I, Patio C, Del Ro J L, et al; Expression of a synthetic gene encoding the sweet-tasting protein thaumatin in the filamentous fungus Penicillium roquefortii, Biotechnology Letters, 1997,19:1185～1191.
Hahm Y T, Batt C A. Expression and Secretion of thaumatin in Aspergillus oryzae, Agric Biol Chem, 1990, 54(10):2513～2520.
Higginbotham J D. Hough C A M. In Sensory properties of foods (Birch G C, Brennan J G, Parker K J,Eds), London. Elsevier Applied Science Press, 1977,pp. 129～149.
Illingworth C, Larson G, Hellekant G. Secretion of the sweet-tasting plant protein thaumatin by Bacillus subtilis,Biotechnol. Lett. 1988, 10:587～592.
Illingworth C, Larson G, Hellekant G. Secretion of the sweet-tasting plant protein thaumatin by Streptomyces lividans,J.,Ind, Microbiol. 1989, 4:37～42.
Kaneko R, Kitabatake N, Sweetness of sweet-protein thaumatin is more thermoresistant under acid conditions than under neutral or alkaline conditions, Biosci Biotechnol Biochem, 2001, 65(2): 409～413.
Kaneko R, Kitabatake N. Structure-sweetness relationship in thaumatin: importance of lysine residues, Chem Senses, 2001, 26:167～177.
Lee J H, Weiekmann J L, Koduri R K, et al; Expression of synthetic thaumatin genes in yeast,Biochemistry, 1988,27:5101～5107.
Lindemann B. Receptors and transduction in taste, Nature, 2001, 413:219～225.
Lyengar K B, Smits P, van der Ouderaa F J, et al; The complete amino acid sequence of the sweet-tasting protein thaumatin I, Eur J Biochem., 1979,96:193～204.
Malepszy S, Szwaeka M, Hrazdina G. Evaluation of transgenic cucumbers expressing the thaumatin gene, Use of agriculturally important genes in biotechnology, Proceeding of the NATO Advanced Research Workshop,Szeged,Hungary, 17-21 October 1999, 2000:131～134.
Margolskee R F. Molecular mechanisms of bitter and sweet taste transduction. J Bio Chem, 2002,277(1): 1～4.
Mitsuhara I, Ugaki M, et al. Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledomous plants. Plant Cell Physiol, 1996,37(1): 49～59.
Moralejo F J, Cardoza R E, Gutierrez S, et al;Thaumatin production in Aspergillus awamori by use of expression cassettes with strong fungal promoters and high gene dosage,Applied and Environmental Microbiology, 1999,65(3): 1168～1174.
Moralejo F J, Cardoza R E, Gutierrez S, et al; Overexpression and lack of degradation of thaumatin in an aspergillopepsin A-defective mutant of Aspergillus awamori containing an insertion in the peA gene, Appl Microbiol Biotechnol, 2000, 54:772～777.
Moralejo F J, Watson A J, ,leenes D J, et al; A defined level of protein disulfide isomerase expression is required for optimal secretion of thaumatin by Aspergillus awamori, Mol Genet Genomics, 2001, 266:246～253.
Slootstra J W, de Geus P, et al; Possible activite site of the sweet-tasting protein thaumatin. Chem Senses,1995,20:535～543.
Suami T, Hough L, Maehinami T, et al; Molecular mechanisms of sweet taste7:The sweet protein, Thaumatin Ⅰ, Food Chemistry, 1997, 60(3): 277～285.
van tier Wel H. Structure-activity relationships in the thaumatin molecule.In "Thaumatin"eds,M Witty, J D Higginbotham,Boca Raton. CRC Press, 1994,pp.115～122.
van der Wel H, Loeve K. Isolation and Characterization of thaumatin Ⅰ and Ⅱ, the sweet-tasting proteins from Thaumatococcus daniellii Benth. Eur J Biochem., 1972,31: 221～225.
Wallroth M, Gerats G M, Rogers S G, et al. Chromosomal localization of foreign genes in Petunia hybrida. Mol Gen Genet. 1986, 202: 146～152.
Witty M. Thaumatin Ⅱ—a palatability protein,Trends in Biotechnology, 1990, 8(5): 113～116.