摘要
乳铁蛋白是一种糖蛋白，为转铁蛋白家族的一员，在人和哺乳动物的许多器官与组织中广泛分布。乳铁蛋白具有多种生物学功能，这些功能包括：促进人体肠道对铁的吸收及调节体内铁的平衡；广谱抗菌(细菌和真菌)、抗病毒感染作用等。把人乳铁蛋白基因转入富含类胡萝卜素和植物纤维的胡萝卜，让它合成具有生物活性的人乳铁蛋白，相当于在营养丰富的胡萝卜中添加新的营养保健因子。乳铁蛋白强的铁螯合能力和广谱抗菌、抗病功能，可提高转基因胡萝卜铁的含量，增强胡萝卜抗菌、抗病能力。
本研究在对胡萝卜组织培养及遗传转化体系进行优化的基础上，构建了人乳铁蛋白基因植物表达载体，在农杆菌介导下，首次把人乳铁蛋白基因导入胡萝卜，获得了转基因植株。对转基因植株进行分子检测，证实了外源基因已整合到胡萝卜基因组中。功能研究和成分测定表明，转基因胡萝卜具有抗菌活性，其铁含量也得到提高。具体结果如下：
1．建立了高效的胡萝卜组织培养及再生体系
以适于生产饮料的胡萝卜“新黑田五寸人参”为材料，研究不同外植体、不同培养基，不同培养条件对胡萝卜愈伤诱导及再生的影响，建立一套高效的胡萝卜组织培养再生体系：最适于诱导愈伤的外植体是弱光或黑暗下发芽7-10d无菌苗下胚轴，最适合的愈伤诱导培养基和继代培养是B_5C(B_5+0.5mg／L 6BA+0.5mg／L2，4-D)，最适于植株再生的培养基为不添加任何激素的B_5或MS，组织培养条件为25℃、光照周期为16hr／8hr。适当照射日光对植株再生具有很好的促进作用。
2．建立了高效的遗传转化体系
以pBI121和pTOK233为转化质粒，在农杆菌LBA4404介导下，对胡萝卜遗传转化体系进行系统研究，首次为该胡萝卜品种建立
一套高效的遗传转化体系，结果为:最适转化受体为新鲜下胚轴
及经预培养的下胚轴;共培养时，低浓度的乙酞丁香酮(25 pM)
对转化具有促进作用;适宜的卡那霉素筛选浓度是loom岁L。筛选
时，卡那霉素浓度先低后高，植株再生时完全去除，有利于缩短
植株再生周期。
3.人乳铁蛋白基因在胡萝卜中的转化及分子检测
以pBll21为中间载体，把人乳铁蛋白基因替换到GUS位置上，
构建成表达载体pBIhLF。在农杆菌LBA4404介导下，把目的基因
导入胡萝卜，获得了外表正常的抗性植株。PCR检测、Southem杂
交证实了外源基因已整合到抗性植株基因组DNA中。对转基因植
株进行RT一PCR检验，得到了阳性结果，说明外源基因在胡萝卜中
能正常转录。
4.转基因植株抗菌能力加强，铁含量得到提高
提取转基因植株叶片粗蛋白，以大肠杆菌DHSQ和酵母菌
AHIOg为试验靶标，侧定了转基因植株粗蛋白的抑菌活性。结果表
明，转基因植株叶片粗蛋白对DH5a有强烈的抑制作用(当培养基
中粗蛋白为lmg/ml时，每ml菌数为对照的1.47%)，对AH109的抑
制作用较弱。按GB汀13883一92标准测定了部分转基因植株叶片中
铁含量。在测定的植株中，铁的含量都得到提高，其中一株提高
64%。
Lactoferrin, a member of transferrin family, is a multifunctional glycoprotein. The biological functions testified include: enhance iron ion assimilation of epithelial cell of intestine and equilibrate body iron concentration; broad spectrum of antiviral activity, antibacterial activity and antifungal activity; modulate marrow cell production and growth; help to mature and regulate a number of immune cells throughout the body, thus boost body immune ability; prevents "free iron" from forming free-radicals; supress tumour growth and prevent tumour formation in animal models. Transformation of human lactoferrin gene to carrot may elevate the transgenic carrot nutritional and functional value, if the transgene expresses correctly, since lactoferrin is a multifunctional protein. In the meantime, high disease resistance and high iron content of the transgenic plants are expected to be achieved due to the natural antibiotic activity and strong iron binding property of the transgenic product.
In the study, sound system of tissue culture and genetic transformation of carrot, cv, Xinheitianwuchunrensheng, are created." A plant expression vector pBIhLF, based on pBin121, was constructed then transferred into carrot. Molecular evidence demonstrated that human lactoferrin gene had been integrated into the genome of transgenic carrot plants. Functional analysis and iron content determination confirm the natural antibiotic activity and iron content of transgenic plants are elevated comparably.
1. Carrot tissue culture and plant regeneration
Factors including explants, medium and culture condition are combined together to study the most efficient protocol of carrot tissue culture and plant regeneration thereof. The most suitable explant is fresh hypocotyls segment and precultured hypocotyls derived from 7-10 day old aseptic plantlets generating in dark or in dim light, the best recipe for cullus induction and subculture is B5C (85 with 0.5mg/L 6BA and 0.5mg/L 2,4-D), the ideal recipe for plant regeneration is 65 or MS free of hormone. A phytotron with a 16/8 h day/night cycle, at 25℃ is feasible for plant regeneration, and occasional exposure to sun light dramatically stimulates plant growth.
2. Establishment of carrot genetic transformation
An efficient transformation protocol based on agrobacterium tumefacien LBA4404 was created in the study. Good results were gained when use fresh hypocotyls as infectious explants, co-cultivate in medium supplemented with low concentration of acetosyringone (25 μ M), screen in medium with lOOmg/L kanamycin. It is time saving when anti-culli are fisrt screened in low concentration of antibiotics then transfered to high concentration ones, and remove antibiotics when regenerate.
3. Transformation of lactoferrin gene to carrot and its molecular evidence
Human lactoferrin gene was incorporated into pBI121 plasmid at Xba I and Sac I sites instead of GUS gene, and then transferred into carrot using agrobacterium-mediated strategy. Transgenic plants were comfirmed by molecular determination including PCR, Southern blot and RT-PCR.
4. Functional analysis and iron content of transgenic plant
Crude proteins of transgenic plant were extracted from leaves, and then used to test its antibiotic activities. In the study, Ecoli DH5a and yeast AH109 were served as target microbes.The growth of Ecoli DH5 a was strongly inhibited by the protein extracts, and yeast was suppressed slightly. Iron content determination show that the iron concentration of transgenic plant is comparable higher (up to 64%) than non-transgenic ones.
引文
郑金贵主编，农产品的品质-营养与保健专辑，福建农业科技1998，增刊
郑金贵，农产品保健品质改良的若干研究动态，福建农业大学学报2000，29(1)：1-6
中国营养学会，中国居民膳食营养素参考摄入量，中国轻工业出版社，2000，15-16
张大兵，张景六，王宗阳，洪孟民．人乳铁蛋白基因在烟草中的表达及抗细菌与病毒的能力．植物生理学报．1999，25(3)：234-243
张大兵，姜育蕾，吴祥甫，洪孟民。人乳铁蛋白基因在昆虫细胞中的表达．生物化学与生物物理学报．1998，30(6)：575-578
王凌健，倪迪安，陈永宁，李忠明，利用转基因胡萝卜表达肺结核疫苗，2001，植物学报，43(2)：132，137．
刘稚，樊梦康，崔，土壤根癌杆菌体外转化胡萝卜悬浮培养细胞，中国科学(B辑)，1987，(5)：507-512．
刘明志，酚类化合物促进含双元载体农杆菌对胡萝卜悬浮细胞的转化和植株再生，植物学报，1996，38(3)：203-208．
李名杨，唐岱，用根癌农杆菌转化获得胡萝卜转化细胞，西南农业大学学报，1989， 11(4)：412-415
Alcantara J., Padda J. S., and Schryvers A. B. The N-linked oligosaccharides of human lactoferrin are not required for binding to bacterial lactoferrin receptors. Canadian Journal of Microbiology, 1992, 38(11):1202-1205,.
Altenbach S.B, Simpson RB, Manipulation of methionine-rich protein genes in plant seeds. Trends Biotechnol 1990, 8:156-160.
Altenbach, S.B. et al. Accumulation of a Brazil nut albumin in seeds of transgenic canola results in enhanced levels of seed protein methionine, Plant Mol. Biol. 1992, 18:235-245
Altenbach, S.B. et al. Enhancement of the methionine content of seed proteins by the expression of a chimeric gene encoding a methionine-rich protein in transgenic plants, Plant Mol. Biol. 1989, 13:513-522
Altenbach, S.B. et al., Cloning and sequence analysis of a cDNA encoding a Brazil nut protein exceptionally rich in methionine, Plant Mol. Biol. 1987, 8:239-250
Andreja Majerle, Jurka Kidri & Roman Jerala. Production of stable isotope enriched antimicrobial peptides in Escherichia coli: An application to the production of a 15N-enriched fragment of lactoferrin, Journal of Biomolecular NMR, 2000,18:145-151
Andrew I. Samuelsen, Ruth C. Martin, David W. S. Mok, and Machteld C. Mok. Expression of the Yeast FRE Genes in Transgenic Tobacco. Plant Physiol. 1998, 118 (1) : 51-58
Anonymous. Engineering plant protein composition for improved nutrition. Trends in plant science. 1998, 3(7) :282-286
Anzai H, Takaiwa F and Katsumata K, Production of Human Lactoferrin in Transgenic Plants. Abstract book of international molecular farming conference, 1999, Canada,
Arondel, V., Lemieux, B., Hwang, I., Gibson, S., Goodman, H.M. and Somerville, C.R., map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis. Science, 1992, 258:1353-1355．
Balestrazzi A, Carbonera D, Cella R. Transformation of Daucus carota hypocotyls mediated by Agrobacterium tumefaciens. J Genet Breed. 1991,45: 135-140．
Bastola R.D and Minocha C.S. Increased polyamines synthesis through transfer of Mouse Ornithine Decarboxylase cDNA in Carrot Promotes Somatic Embryogenesis. Plant Physiol., 1995,109:63-71．
Beach LR, Ballo B, Enhancing the nutritional value of seed crops. In Biosynrhesis and molecular regulation of amino acids in plants. Edited by Singh BK, Flares HE, Shannon JC. Rockville: American Society of Plant Physiologists; 1992, 229-238．
Beebe S, Gonzalez AV, Rengifo J: Research on trace minerals in the common bean. Food Nutr Bull 2000, 21:387-391．
Ben Miflin, Johnathan Napier and Peter Shewry. Improving plant product quality, Nature biotech 1999, 17:13s-14s
Benfey PN, Ren L, Chua NH. The CaMV 35S enhancer contains at least two domains which can confer different developmentaland tissue-specific expression patterns. EMBO J, 1989, 8:2195-2202．
Bezault J. A., Bhimani R., Wiprovnick J., and Furmanski P., Human lactoferrin inhibits growth of solid tumors and development of experimental metastases in mice. Cancer Research Baltimore, 1994, 54(9) :2310-2312,.
Bhimani RS, Vendrov Y, Furmanski P. Influence of lactoferrin feeding and injection against systemic staphylococcal infections in mice. J Appl Microbiol 1999, 86(1) : 135-44．
Blakeborough.P; Salter.DN, Zinc binding by lactoferrin and human milk. Trace elements in man and animals : TEMA 5 : proceedings of the fifth International Symposium on Trace Elements in Man and Animals / editors C.F. Mills, I. Bremner, & J.K. Chesters. Farnham Royal, Slough : Commonwealth Agricultural Bureaux, 1985, 420-423．
Boddupalli, S.S., Stark, D.M., Barry, G.F. and Kishore, G.M. Effect of overexpressing ADPGIc pyrophosphorylase on the oil biosynthesis in
canola. Abstracts, NPLC Symposium on Biochemistry and Molecular Biology of Plant Fatty Acids and Glycerolipids, 1995． 42．
Bolton GW. Nester EW, Gordon MP. Plant phenolic compounds induce expression of the Agrobacterium tumefaciens loci needed for virulence. Science. 1986, 232:983-983．
Brown, A.P., Brough, C.L., Kroon, J.T.M. and Slabas, A.R. Identification of a cDNA that encodes a 1-acyl-sn-glycerol-3-phosphate acyltransferase from Limnanthes douglasii. Plant Molecular Biology, 1995, 29(2) :267-278．
Browse, J. and Somerville, C. Glycerolipid metabolism, biochemistry and regulation. Annual Review of Plant Physiology and Plant Molecular Biology, 1991,42:467-506．
Cahoon EB, Lindqvist Y, Schneider G, Shanklin J. Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position. Proc Natl Acad Sci USA 1997, 94:4872-4877
Cahoon, E.B. and Shanklin, J. Engineering of the chain-length and double bond positional specificities of acyl-ACP desaturases. Abstracts, NPLC Symposium on Biochemistry and Molecular Biology of Plant Fatty Acids and Glycerolipids. 1995．
Cahoon, E.B., Shanklin, J. and Ohlrogge, J.B.. Expression of a novel coriander desaturase results in petroselinic acid production in transgenic tobacco. Proceedings of the National Academy of Sciences, USA, 1992, 89:11184-11188．
Cangelosi GA, Martinetti G, Leigh JA. Role of Agrobacterium tumefaciens ChvA protein in export of β-1． 2-glucan. J bacterial. 1989, 171:1609-1615．
Cases S, Smith SJ, Zheng Y, Myers HM, Sande ER, Novak S, Lear SR, Erickson SK, Farese RV. Cloning and expression of a candidate gene for diacylglycerol acyltransferase. FASEB J 1998,112:A814
Chui, C-F. and Falco, S.C. A new methionine-rich seed storage protein from maize, Plant Physiol. 1995, 107: 291
Daniel K.X. Chong & William H.R. Langridge. Expression of full-length bioactive antimicrobial human lactoferrin in potato plants. Transgenic Research. 2000, 9: 71-78．
Defer MC, Dugas B, Picard O, Damais C. Impairment of circulating lactoferrin in HIV-1 infection. Cell Mol Biol (Noisy-le-grand) 1995 May;41(3) :417-21．
DellaPenna D, Nutritional Genomics: Manipulating Plant Micronutrients to Improve Human Health, SCIENCE 1999, 285(16) : 375-379
DellaPenna D. Plant Metabolic Engineering. Plant Physiol., 2001, 125(1) :160-163,.
Dial EJ, Hall LR, Serna H, Romero JJ, Fox JG, Lichtenberger LM. Antibiotic properties of bovine lactoferrin on Helicobacter pylori. Dig Dis
Sci, 1998, 43(12) :2750-6．
Dormann, P., Spener, F. and Ohlrogge, J.B.. Characterization of two acyl-ACP thioesterases specific for medium chain acyl-ACP and oleoyl-ACP from developing Cuphea seeds. Planta, 1993, 189:425-432
Dormann, P., Voelker, T.A. and Ohlrogge, J.B.. Cloning and expression in Escherichia coli of a novel thioesterase from Arabidopsis thaliana specific for long chain acyl-acyl carrier proteins. Archives of Biochemistry and Biophysics, 1994, 316:612-618．
Dybing, C.D. and Craig, B.M.. Fatty Acid biosynthesis and incorporation into lipid classes in seed and seed tissues of flax. Lipids, 1970, 5:422-429．
Eccleston VS, Ohlrogge JB. Expression of lauroyl-acyl carrier protein thioesterase in Brassica napus seeds induces pathways for both fatty acid oxidation and biosynthesis and implies a set point for triacylglycerol accumulation. Plant Cell 1998, 10:613-621
Eda S., Kikugawa K., and Beppu M.. Binding characteristics of human lactoferrin to the human monocytic leukemia cell line THP-1 differentiated into macrophages. Biological and Pharmaceutical Bulletin, 1996, 19 (2) : 167-175．
Eide D., Broderius M., Fett J, and Guerinot L.M.. A novel iron-regulated metal transporter from plants identified by functional expression in yeast. PNAS, 1996, 93 (11) :5624-5628,.
Falco SC, Beaman C, Chui CF, Guida T, Hirata L, Jones T.Keeler S, Knowlton S, Kostow C, Locke M et al., Transgeniccrops with improved amino acid composition. J Cell Biochem 1994, 181(suppl A):79．
Falco, S.C. et al. Transgenic canola and soybean seeds with increased lysine, Biotechnology, 1995 ,13:577-582
Fox, B.G., Shanldin, J., Somerville, C. and Munck, E.. stearoyl-acyl carrier protein δ 9 desaturase from Ricinus communis is a diioron-oxo protein. Proceedings of the National Academy of Sciences, 1993, 90:2486-2490．
Fuller, M.F. et al. The optimum dietary amino acid pattern for growing pigs, Br.J.Nutri. 1989,62: 255-267
Fumiyuki Goto and Toshihiro Yoshihara, Improvement of micronutrient contents by genetic engineering-development of high iron conten crops,Plant biotechnology, 2001, 18(1) :7-15
Galili G. Regulation of Lysine and Threonine Synthesis. Plant Cell, 1995, 7 (7) :899-906
Gilbert M.O., Zhang Y.Y., Punja Z.K., Introduction and expression of chitinase encoding genes in carrots following Agrobacterium-mediated transformation, In Vitro Cell, 1996,32: 171-178．
Gogarten JP, Fichmann J, BraunY, Morgan L, Styles P, DeLapp K, Taiz L. The use of antisense mRNA to inhibit the tonoplast H+ ATPase in carrot.
Plant Cell, 1992,4:851-864．
Goodman R. E. and Schanbacher F. L. Bovine lactoferrin mRNA: sequence, analysis, and expression in the mammary gland. Biochemical and Biophysical Research Communications, 1991, 180 (1) :75-84．
Goto F, Yoshihara T, Shigemoto N, Toki S, Takaiwa F, Iron fortification of rice seed by the soybean ferritin gene, Nature biotech 1999,117:282-286
Gregorio B. G, Progress in breeding for trace minerals in staple crops, J. Nutr. 2002, 132:500s-502s
Gregorio GB, Senadhira D, Htut H, Graham RD: Breeding for trace mineral density in rice. Food Nutr Bull, 2000, 21:382-386．
Griffiths, G. and Harwood, J.L. The regulation of triacylglycerol biosynthesis in cocoa (Theobroma cacao) L. Planta, 1991, 184:279-284．
Gunstone, F.D., Harwood, J.L. and Padley, F.B.. The Lipid Handbook. London: Chapman Hall, 1994．
Habben E J and Larkins A B, Genetic modification of seed proteins, current opinion in biotechnology, 1995, 6:171-174
Hadama T, Nishiuchi T, kodama H, et al. cDNA cloning of a wounding-inducible gene encoding a plastid w3 fatty acid desaturase from tobacco. Plant Cell Physiol, 1996, 37(5) :606-611
Harmsen MC, Swart PJ, de Bethune MP, Pauwels R, De Clercq E, The TH, Meijer DK. Antiviral effects of plasma and milk proteins: lactoferrin shows potent activity against both human immunodeficiency virus and human cytomegalovirus replication in vitro. J Infect Dis, 1995,172(2) :380-8．
Hawkins DJ, Kridl JC. Characterization of acyl-ACP thioesterases of mangosteen(Garcinia manostana) seed and high levels of stearate production in transgenic canola. Plant J, 1998,13:743-752
Higgins, TJV, Synthesis and regulation of major proteins in seeds. Annu Rev Plant physiol, 1984, 35:191-221．
Hoffman LM, Donaldson DD, Herman EM. A modified storage protein is synthesized, processed, and degraded in the seeds of transgenic plants. Plant Mol Biol, 1988,11:717-729．
Hole CC: Carrots. In: Zamski E, Schaffer AA (eds). Photoassimilate distribution in Plants and Crops, Marcel Dekker, New York, 1996． 671-690．
Horiguchi G, Iwakawa H, Kodama H, et al. Expression of gene for plastid w3 fatty acid desaturase and changes in lipid and fatty acid compositions in light-and dark-grown wheat leaves. Physiol Plant, 1996, 96:275-283
Humphrey D. B., Huang N. and Klasing C. K., Rice Expressing Lactoferrin and Lysozyme Has Antibiotic-Like Properties When Fed to Chicks, J Nutr 2002; 132(6) : 1214-8
Idriss E. E, Makarewicz O., Farouk A., Rosner K., Greiner R., Bochow H., Richter T., and Borriss R., Extracellular phytase activity of Bacillus amyloliquefaciens F2B45 contributes to its plant-growth-promoting effect. Microbiology 2002, 148 (7) :2097-2109,.
Ishizaki O.I., Nishida O.I., Agata K., Eguche G. and Murata N., Cloning and nucleotide sequence of cDNA for the plastid glycerol-3-phosphate acyltransferase from squash. Federation of European Biochemical Societies, 1988, 238:424-430．
James, Jr., D.W., Lira, E., Keller, J., Plooy, I., Ralston, E. and Dooner, H.K.. Directed tagging of the Arabidopsis fatty acid elongation 1 (FAE1) gene with the maize transposon activator. The Plant Cell, 1995, 7:309-319．
Joseph Hirschberg, Production of high-value compounds: carotenoids and vitamin E, Current opinion in biotechnology 1999, 10:186-191
Karchi H., Shaul O., and Galili G. Lysine Synthesis and Catabolism are Coordinately Regulated During Tobacco Seed Development. PNAS, 1994, 91 (7) :2577-2581．
Karchi, H., Shaul, O. and Galili, G. Seedspecific expression of a bacterial desensitized aspartate kinase increases the production of seed threonine and methionine in transgenic tobacco, Plant J., 1993 ,3: 721-727
Khan, M.R.I, et al. Accumulation of a sulphur-rich seed albumin from sunflower in the leaves of transgenic subterranean clover (Trifolium subterraneum L), Transgenic Res. 1996, 5:179-185
Kinney A. J. Genetic modification of the storage lipids of plants. Current Opinions Biotechnology, 1994, 5:144-151．
Kirihara, J.A., Petri, J.B. and Messing, J., Isolation and sequence of a gene encoding a methionine-rich 10-kDa protein from maize, Gene, 1988, 71:359-370
Kishore M. G. and Shewmaker C. Biotechnology: Enhancing human nutrition in developing and developed worlds. PNA, S 1999, 96 (11) :5968-5972
Kjemtrup S, Herman EM, Chrispeels Ml, Correct post-translational modification and stable vacuolar accumulation of phytohemagglutinin engineered to contain multiple methionine residues. Eur J Biochem 1994, 226:385-391．
Klein, B., Pawlowski, K., Horicke-Grandpierre, C., Schell, J. and Topfer, R. Isolation and characterization of a cDNA from Cuphea lanceolata encoding a,b-ketoacyl-ACP reductase. Molecular and General Genetics, 1992,233:122-128．
Knutzon, D.S., Lardizabal, K.D., Nelsen, J.S., Bleibaum, J.L., Davies, G.A. and Metz, J. cloning of a coconut endosperm cDNA encoding a 1-acyl sn-gylcerol-3-phosphate acyltrafisferase which accepts medium chain fatty acids. Plant Physiology, 1995, 109:999-106．
Knutzon, D.S., Thomson, G.A., Radke, S.E. and Johnson, W.B., Modification of Brassica seed oil by antisense expression of a steroyl-acyl carrier protein desaturase gene. Proceedings of the National Academy-of Sciences, 1992, 89: 2624-2628
Kortt, A.A. et al,. Amino acid and cDNA sequence of a methionine-rich 2S protein from sunflower seed (Helianthus annuus L), Eur. J. Biochem. 1991, 195:329-334
Krimpenfort P., The production of human lactoferrin in the milk of transgenic animals. Cancer Detection and Prevention, 1993, 17(2) :301-305
Kruzel ML, Harari Y, Chen CY, Castro GA. The gut. A key metabolic organ protected by lactoferrin during experimental systemic inflammation in mice. Adv Exp Med Biol, 1998, 443:167-73
Kuwata H, Yip TT, Tomita M, Hutchens TW. Direct evidence of the generation in human stomach of an antimicrobial peptide domain (lactoferricin) from ingested lactoferrin. Biochim Biophys Acta 1998, 1429(1) :129-41
L. Davidsson, P. Kastenmayer, M. Yuen, B. Lonnerdal, and R. F. Hurrell. Influence of lactoferrin on iron absorption from human milk in infants. Pediatric Research, 1994, 35 (1) :117-124
L. J. Alexander, W. B. Levine, C. T. Teng, and C. W. Beattie. Cloning and sequencing of the porcine lactoferrin cDNA. Animal Genetics, 1992, 23(3) :251-256
Lee WJ, Farmer JL, Hilty M, Kim YB. The Protective Effects of Lactoferrin Feeding against Endotoxin Lethal Shock in Germfree Piglets. Infect Immun Apr. 1999, 66(4) :1421-1426
Leonard JM, Knapp SJ, Slabaugh MB. A Cuphea B-ketoacyl-ACP synthase shifts the synthesis of fatty acids towards shorter chains in Arabidopsis seeds expressing Cuphea FatB thioesterases. Plant J, 1998, 13:621-6
Liang Q. and Richardson T.. Expression and characterization of human lactoferrin in yeast Saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry, 1993,41 (10) :1800-1807
Liang Q., Jimenez R. Flores, and Richardson T. Screening and cloning a cDNA coding for lactoferrin from human mammary gland. Journal of Dairy Science, 1989, 72 (Suppl. 1) :154．
Liang Q., Jimenez R. Flores, and Richardson T.. Cloning two cDNA sequences coding for lactoferrin from human mammary gland. Journal of Dairy Science, 1990, 73 (Supplement 1) :107
Lighter J, Wu J, Browse J. A mutant of Arabidopsis with increased level of stearic acid. Plant Physiol, 1994, 106:1443-1451
Lisa Molvig, Linda M. Tabe, Bjorn O. Eggum, Andrew E. Moore, Stuart Craig, Donald Spencer, and Thomas J. Higgins. Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene. PNAS, 1997, 94(16) :8393-8398．
Lonnerdal B, Trace element absorption in infants as a foundation to setting upper limits for trace elements in infant formulas, Journal-of-Nutrition, 1989,119(12S): 1839-1845
Lonnerdal B. Expression of Human Milk Proteins in Plants. J Am Coll Nutr, 2002,21:218S.
M. S. Cohen, J. Mao, G. T. Rasmussen, J. S. Serody, and B. E. Britigan. Interaction of lactoferrin and lipopolysaccharide (LPS): effects on the antioxidant property of lactoferrin and the ability of LPS to prime human neutrophils for enhanced superoxide formation. Journal of Infectious Diseases , 1992, 166 (6) :1375-1378．
M. T. Ruel and H. E. Bouis. Plant breeding: a long-term strategy for the control of zinc deficiency in vulnerable populations. Am J Clin Nutr, 1998, 68 (2) :488S-494．
M. W. Rey, S. L. Woloshuk, H. A. DeBoer, and F. R. Pieper. Complete nucleotide sequence of human mammary gland lactoferrin. Nucleic Acids Research, 1990, 18 (17) :5288．
Magnuson J. S., Henry J. F., Yip T. T., and Hutchens T. W.. Structural homology of human, bovine, and porcine milk lactoferrins: evidence for shared antigenic determinants. Pediatric Research, 1990, 28 (2) :176-181
Maneva, B. Taleva, V. Manev, and L. Sirakov. Lactoferrin binding to human platelets. International Journal of Biochemistry, 1993, 25 (5) :707-712,.
Markus Hardegger and Arnd Sturm. Transformation and regeneration of carrot (Daucus carota L.). Molecular Breeding, 1998, 4:119-127．
Mead P. E. and Tweedie J. W.. cDNA and protein sequence of bovine lactoferrin. Nucleic Acids Research, 1990,18 (23) :7167．
Mertz ET, Bates LS, Nelson OE, Mutant gene that changes protein composition and increases lysine content of maize endosperm. Science 1964, 145:279-280．
Michael A. Grusak and Dean Dellapenna, Improving the nutrient composition of plants to enhance human nutrition and health, Plant Mol Biol 1999, 50:133-161
Michael B Zimmermann and Richard F Hurrell, Improving iron, zinc and vitamin A nutrition through plant biotechnology, Current Opinion in Biotechnology, 2002,13:142-145
Michaela Zeh, Anna Paola Casazza, Oliver Kreft, Ute Roessner, Katrin Bieberich, Lothar Willmitzer, Rainer Hoefgen, and Holger Hesse.
Antisense Inhibition of Threonine Synthase Leads to High Methionine Content in Transgenic Potato Plants. Plant Physiol. 2001,127(3) :792-802
Mitra A. and Zhang Z. Y. Expression of a human lactoferrin cDNA in tobacco cells produces antibacterial protein(s). Plant Physiology, 1994, 106(3) :977-981
Molenaar J., Davis S. R., and Wilkins R. J.. Expression of alpha-lactalbumin, alpha-S1-casein, and lactoferrin genes is heterogeneous in sheep and cattle mammary tissue. Journal of Histochemistry and Cytochemistry, 1992, 40 (5) :611-618,.
Molvig, L. et al. Enhanced methionine levels and increased nutritive value of seeds of transgenic lupins (Lupinus angustifolius L.) expressing a sunflower seed albumin gene, Proc. Natl. Acad. Sci. 1997, 94: 8393-8398
Murphy DJ. engineering oil production in rapeseed and other oil crops, trends biotechnol, 1996,14:206-213
Murphy, D.J. (1994) . Designer Oil Crops. VCH, Weinheim. Murphy, D.J:. Structure, function and biogenesis of storage lipid bodies and oleosins in Plants. Progress in Lipid Research, 1993, 32:247-280．
Natasha Crotz and Mary Lou Guerinot, Limiting nutrients: and old problem with new solutions? Current opinion in plant biology, 2002, 5:158-163
Nelson OE: Genetic modification of protein quality in plants. Adv Agron 1969,21:171-194．
Newton IS, Long-chain polyunsaturated fatty acids-the new frontier in nutrition. Lipid Technol, 1998, 10:77-81
Nikawa H., Samaranayake L. P., Tenovuo J., Pang K. M., and Hamada T.. The fungicidal effect of human lactoferrin on Candida albicans and Candida krusei. Archives of Oral Biology, 1993, 38 (12) : 1057-1063,.
Nishiuchi T, Nakamura T, Abe T, et al. Tissue-specific and light responsive regulation of the promoter region of the Arabdopsis thiana chloroplast w3 fatty acid desaturase gene. Plant Mol Biol, 1995, 29:599-609
Nordlee, J.A. et al. Identification of a Brazil-nut allergen in transgenic soybeans, New Engl. J. Med. 1996, 334: 688-692
Nuijens H J., Patrick H. C. van Berkel, Marlieke E. J. Geerts, Peter Paul Hartevelt, Herman A. de Boer, Harry A. van Veen, and Frank R. Pieper. Characterization of Recombinant Human Lactoferrin Secreted in Milk of Transgenic Mice. THE JOURNAL OF BIOLOGICAL CHEMISTRY . 1997, 272(13) :8802-8807,
Nuijens J. H., Berkel van PHC, Geerts M. E. J., Hartevelt P. P., Boer de HA, Veen van HA, Pieper F. R., Berkel Van P. H. C., Characterization of recombinant human lactoferrin secreted in milk of transgenic mice. J.Biol.Chem. 1997, 272 (13) :8802-8807
Nuijens J. H., PHC van Berkel, M. E. J. Geerts, P. P. Hartevelt, HA de Boer, HA van Veen, F. R. Pieper, P. H. C. Van Berkel, H. A. De Boer, and H. A. Van Veen. Characterization of recombinant human lactoferrin secreted in milk of transgenic mice. J.Biol.Chem., 1997, 272(13) :8802-8807．
Ohlrogge J and Browse J., Lipid Biosynthesis The Plant Cell, 1995, 7: 957-970,
Ohlrogge JB, Design of new plant products: engineering of fatty acid metabolism. Plant Physiol, 1994, 104:821-826
Okuley, J., Lightner, J., Feldmann, K., Yadav, N., Lark, E. and Browse, J. The Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell, 1994, 6:147-158．
Olga Sayanova, Mark A. Smith, Peter Lapinskas, A. Keith Stobart, Gary Dobson, William W. Christie, Peter R. Shewry, and Johnathan A. Napier. Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of Delta 6-desaturated fatty acids in transgenic tobacco. PNAS , 1997, 94(8) :4211-4216
Orla M. Conneely. Antiinflammatory Activities of Lactoferrin. J Am Coll Nutr,2001, 20 (90005) :389S-395．
Paola Lucca, Richard Hurrell, and Ingo Potrykus, Fighting Iron Deficiency Anemia with Iron-Rich Rice, Journal of the American College of Nutrition, 2002, 21(3) :184S-190S
Patrick H.C. van Berkel, Mick M. Welling, Marlieke Geerts, Harry A. van Veen, Bep Ravensbergen, Mourad Salaheddine, Ernest K. J. Pauwels, Frank Pieped, Jan H. Nuijens, and Peter H, Nibbering. Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nature biotechnology. 2002, 20:484-487
Pawlicki N., Rajbir S., Sangwan B., Sangwan-Norreel S., Factors influencing the Agrobacterium tumefaciens-mediated transformation of carrot (Daucus carota L.), Plant Cell Tissue Organ Culture, 1992, 31:129-139．
Percival M. Intestinal Health. Clin. Nutri. Insights. 1997, Vol 5． No 5,1-6．
Pierce, D. Colavizza, M. Benaissa, P. Maes, A. Tartar, J. Montreuil, and G. Spik. Molecular cloning and sequence analysis of bovine lactotransferrin. European Journal of Biochemistry, 1991,196 (1) :177-184．
Platenburg G. J., Kootwijk E. P. A., Kooiman P. M., Woloshuk S. L, Nuijens J. H., Krimpenfort P. J. A., Pieper F. R., Boer de HA, Strijker R., and Boer De H. A. Expression of human lactoferrin in milk of transgenic mice. Transgenic Research, 1994, 3 (2) :99-108,.
Postaire E. Evaluation of the antiradical protector effect of multifermented milk serum with reiterated dosage in rats. C R Seances Soc Biol Fil 1995; 189(6) : 1191-7．
Potrykus I. Golden Rice and Beyond. Plant Physiol. 2001, 125(3) : 1157-1161,.
Provost F. le, M. Nocart, G. Guerin, P. Martin, and F. Le Provost. Characterization of the goat lactoferrin cDNA: assignment of the relevant locus to bovine U12 synteny group. Biochemical and Biophysical Research Communications, 1994, 203 (2) :1324-1332
Puddu P, Borghi P, Gessani S, Valenti P, Belardelli F, Seganti L. Antiviral effect of bovine lactoferrin saturated with metal ions on early steps of human immunodeficiency virus type 1 infection. Int J Biochem Cell Biol, 1998, 30(9) : 1055-62．
Radwanski E. R. and Last R. L. Tryptophan Biosynthesis and Metabolism: Biochemical and Molecular Genetics. Plant Cell, 1995, 7(7) :921-934,.
Reddy A S, Muccio M I, Gross L M, et al. Isolation of A6 desaturase gene from the cyanobacterium Synechocystis sp. strain PCC 6803 by gain-of-function expression in Anabaenasp. strain. Plant Mol Biol, 1993, 27:293-300
Rice JA, Keeler SJ, Hirata LT, Beaman C, Webber P, Jones T, McAdams S, Falco SC, Expression of synthetic high lysine seed storage proteins can significantly increase the accumulated levels of lysine in mature seeds of transgenic crop plants. J Cell Biochem 1994,18(suppl A):107．
Saalbach, G. et al. Stable expression of vicilin from Vicia faba with eight additional single methionine residues but failure of accumulation of legumin with an attached peptide segment in tobacco seeds, Mol. Breed., 1995, 1:245-258
Saalbach, I. et al. The sulphur-rich Brazil nut 2S albumin is specifically formed in transgenic seeds of the grain legume Vicia narbonensis, Euphytica. 1995, 85:181-192
Saalback I, Pickardt T, Machemehl F, Saalbach C, Schieder 0, Miintz K. A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Berthofletia excefsa H.B.K.) is stably expressed and inherited in transgenic grain legumes. Mol Gen Genet 1994, 242:226-236．
Sakamoto N. Antitumor effect of human lactoferrin against newly established human pancreatic cancer cell line SPA. Gan To Kagaku Ryoho, 1998, 25(10) : 1557-63．
Salmon V, Legrand D, Slomianny MC, el Yazidi I, Spik G, Gruber V, Bournat P, Olagnier B, Mison D, Theisen M, Merot B., Production of human lactoferrin in transgenic tobacco plants. Protein Expr Purif 1998, 13(1) : 127-35
Samyn-Petit B, Gruber V, Flahaut C, Wajda-Dubos JP, Farrer S, Pons A, Desmaizieres G, Slomianny MC, Theisen M, Delannoy P. N-glycosylation potential of maize: The human lactoferrin used as a model. Glycoconj J, 2001, 18(7) :519-27
Sang H. L, Boer de HA, and Boer De H. A.. Production of biomedical proteins in the milk of transgenic dairy cows: the state of the art. Journal of Controlled Release, 1994, 29(3) :213-221,.
Sayanova O, Smith MA, Lapinskas P, Stobart AK, Dobson G, Christie WW, Shewry PR, Napier J. Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of delta-6-desaturated fatty acids in transgenic tobacco. Proc Natl Acad Sci USA, 1997, 94:4211-4216
Scott R, Draper J. Transformation of carrot tissues derived from proembryogenic suspension cells: a useful model system for gene expression studies in plants. Plant Mol Biol, 1987, 8: 265-274．
Seyfert H. M., Tuckoricz A., Interthal H., Koczan D., and Hobom G.. Structure of the bovine lactoferrin-encoding gene and its promoter. Gene, 1994, 143(2) :265-269,.
Shaver M. J, Bittel C D., Sellner M. J, Frisch A. D, Somers A D., and Gengenbach G B., Single-amino acid substitutions eliminate lysine inhibition of maize dihydrodipicolinate synthase. PNAS, 1996, 93 (5) : 1962-1966,.
Sheikholeslam SN.Weeks DP. Acetosyringone promotes high efficiency transformation of Agrobacterium thaliana explants by Agrobacterium tumefaciens. Plant Mol Biol, 1987, 8: 291-298．
Shinmoto H., S. Dosako, and I. Nakajima. Anti-oxidant activity of bovine lactoferrin on iron/ascorbate induced lipid peroxidation. Bioscience, Biotechnology and Biochemistry, 1992, 56(12) :2079-2080．
Shintani D and DellaPenna D. Elevating the Vitamin E Content of Plants Through Metabolic Engineering. Science, 19982, 82 (5396) :2098-2100,.
Shirsat N. V., Bittenbender S., Kreider B. L., and Rovera G.. Structure of the murine lactotransferrin gene is similar to the structure of other transferrin-encoding genes and shares a putative regulatory region with the murine myeloperoxidase gene. Gene, 1992,110 (2) :229-234
Slabaugh MB, Leonard JM, Knapp SJ. Condensing enzymes from Cuphea wrightii associated with medium chain fatty acid biosynthesis. Plant J, 1998, 13:611-620
Soheil S. Mahmoud and Rodney B. Croteau. Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase. Proc. Natl. Acad. Sci. USA, 2001, 98(15) : 8915-8920
Somerville C, Browse J. Dissecting desaturation. Trends in Cell Biol,1996, 6:148-153
Stachel SE, Messens E. van montaga M et al. identification of single molecular produced by wounded plant cells that activated T-DNA transfer in Agrobacterium tumefaciens. Nature. 1985, 318:624-629．
Striiker R., EyestoneW., Kooiman P., Kootwijk E., Krimpenfort P., Nuyens J., Pieper F., Platenburg G., Rademakers A., Woloshuk S., de Boer H., and Boer De H., Expression of biomedical proteins in milk. Proceedings European Congress on Biotechnology, 1990, 5 (2) :949-952．
Subhra Chakraborty, Niranjan Chakraborty, and Asis Datta. Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus. PNAS, 2000, 97(7) :3724-3729．
SunJung Kim, KoWoon Lee, DaeYeul Yu, YongMahn Han, ChulSang Lee, MyungSoo Nam, HyungBae Moon, KyungKwang Lee, S. J. Kim, K. W. Lee, D. Y. Yu, Y. M. Han, C. S. Lee, M. S. Nam, H. B. Moon, and K. K. Lee. Expression analysis of a bovine beta-casein/human lactoferrin hybrid gene in transgenic mice. Journal of Reproduction and Development, 1997, 43(2) :143-149．
SunJung Kim, KoWoon Lee, DaeYeul Yu, YongMahn Han, ChulSang Lee, MyungSoo Nam, HyungBae Moon, KyungKwang Lee, S. J. Kim, K. W. Lee, D. Y. Yu, Y. M. Han, C. S. Lee, M. S. Nam, H. B. Moon, and K. K. Lee. Expression analysis of a bovine beta-casein/human lactoferrin hybrid gene in transgenic mice. Journal of Reproduction and Development, 1997, 43 (2) :143-149
Superti F, Ammendolia MG, Valenti P, Seganti L. Antirotaviral activity of milk proteins: lactoferrin prevents rotavirus infection in the enterocyte-like cell line HT-29． Med Microbiol Immunol (Berl) 1997, 186(2-3) :83-91．
Swart PJ, Kuipers EM, Smit C, Van Der Strate BW, Harmsen MC, Meijer DK. Lactoferrin. Antiviral activity of lactoferrin. Adv Exp Med Biol, 1998,443:205-13．
T. W. Okita, Y. S. Hwang, J. Hnilo, W. T. Kim, A. P. Aryan, R. Larson, and H. B. Krishnan. Structure and expression of the rice glutelin multigene family. J.Biol.Chem., 1989, 264 (21) :12573-12581
Tabe LM, Higgins CM, McNabb WC, Higgins TJV: Genetic engineering of grain and pasture legumes for improved nutritive value. Genetica, 1993, 90:191-200．
Takaichi M., Oeda K., Transgenic carrots with enhanced resistance against two major pathogens, Erysiphe heraclei and Alternaria dauci, Plant Science, 2000,153:135-144,
Tang GQ and Sturm A, Antisense repression of sucrose synthase in carrot (Daucus carota L.) affects growth rather than sucrose partitioning. Plant Molecular Biology. 1999, 41: 465-479,.
Thomas J, Guiltinan M, Bustos S, Thomas T, Nessler C. Carrot (Daucus carota) hypocotyl transformation using Agrobacterium tumefaciens. Plant Cell Rep, 1989, 8:354-357．
Thomas, T.L., Nunberg, A., Reddy, A.S., Nuccio, M.L. and Beremand, P. (1995) . Cloning and expression of A 6-desaturase genes in transgenic planks. Abstract, National Plant Lipid Cooperative Symposium, South Lake Tahoe, CA, 1995, June 1-4．
Tokuji, Y; Fukuda, H. A., rapid method for transformation of carrot (Daucus carota L.) by using direct somatic embryogenesis.Bioscience. Biotechnology and Biochemistry, 1999, 63:519-523．
Tonnet, M.L. and Green, A.G. Characterization of the seed and leaf lipids of high and low linolenic acid flax genotypes. Archives of Biochemistry and Biophysics, 1987, 252:646-654．
Topfer, R. and Martini, N. Molecular cloning of cDNAs or genes encoding proteins involved in de novo fatty acid biosynthesis in plants. Plant Molecular Biology, 1994, 143:416-425．
Tsuda H, Sekine K, Nakamura J, Ushida Y, Kuhara T, Takasuka N, Kim DJ, Asamoto M, Baba-Toriyama H, Moore MA, Nishino H, Kakizoe T. Inhibition of azoxymethane initiated colon tumor and aberrant crypt foci development by bovine lactoferrin administration in F344 rats. Adv Exp Med Biol, 1998, 443:273-84．
Ushida Y, Sekine K, Kuhara T, Takasuka N, ligo M, Tsuda H. Inhibitory effects of bovine lactoferrin on intestinal polyposis in the Apc(Min) mouse. Cancer Lett, 1998, 134(2) :141-5．
V. Salmon, D. Legrand, B. Georges, M. C. Slomianny, B. Coddeville, and G. Spik. Characterization of human lactoferrin produced in the baculovirus expression system. Protein Expression and Purification, 1997, 9(2) :203-210
Voelker, T.A., Worrell, A.C., Anderson, L., Bleibaum, J., Fan, C., Hawkins, D.J., Radke, S.E. and Davies, H.M. Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants. Science, 1992, 257:72-73．
Ward P. P., Lo J. Y., Duke M., May G. S., Headon D. R., and Conneely O. M.. Production of biologically active recombinant human lactoferrin in Aspergillus oryzae. Bio Technology, 1992, 10 (7) :784-789．
Ward P. P., Piddington C. S., Cunningham G. A., Zhou X., Wyatt R. D., and Conneely O. M., A system for production of commercial quantities of human lactoferrin: a broad spectrum natural antibiotic. Bio Technology, 1995, 13(5) :498-502．
Wayne R. Bidlack. Lactoferrin: Natural, Multifunctional, Antimicrobial. J Am Coll Nutr, 2001, 20 (4) :343-347
Wurtele E., Bulka K., A simple, efficient method for the Agrobacterium-mediated transformation of carrot callus cells, Plant Sci., 1989,61:253-262．
Xudong Ye, Salim Al Babili, Andreas ti, Jing Zhang, Paola Lucca, Peter Beyer, and Ingo Potrykus. Engineering the Provitamin A (-Carotene)
Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm. Science , 2000, 287 (5451 ):303-305．
Yoo YC, Watanabe S, Watanabe R, Hata K, Shimazaki K, Azuma I. Bovine lactoferrin and lactoferricin, a peptide derived from bovine lactoferrin, inhibit tumor metastasis in mice. Jpn J Cancer Res, 1997, 88(2) : 184-90．
Yuan, L, Voelker, T.A. and Hawkins, D.J. Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering. Proceedings of the National Academy of Sciences, USA, 1995,92, 10639-10643．
Yukawa Y, Takaiwa F, Shojik K, et al. Structure and expression of two seed-specific cDNA encoding stearoyl-acyl carrier protein desaturase from sesame, Sesamum indicum J. Plant Cell Physiol, 1996, 37(2) :201-205
Zhang GH, Mann DM, Tsai CM. Neutralization of endotoxin in vitro and in vivo by a human lactoferrin-derived peptide. Infect Immun 1999 , 67(3) : 1353-8．
Zhang Z.; Goyne D.P.; Vidaver A.K.; Mitra A. Expression of human lactoferrin cDNA confers resistance to Ralstonia solanacearum in transgenic tobacco plants. Phytopathology, 1998, 88 (7) :730-734
Zou J, Katavic V, Giblin EM, Barton DL, MacKenzie SL, Keller WA, Hu X, Taylor DC. Modification of seed oil content and acyl composition in the Brassicaceae by expression of a yeast sn-2 acyltransferase gene. Plant Cell, 1997,9:909-923
