Expression And Bioactivity Studies Of Human Lactoferrin In Transgenic Carrot (Daucus Carota L.)

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 regenerationFactors 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 transformationAn 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 evidenceHuman 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 plantCrude 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.