Study On Genetic Transformation Of Human-lactoferrin Gene To Tomato

Lactoferrin, a member of transferrin family, is a multifunctional glycoprotein. The biological functions testified include: enhance iron 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; prevent "free iron" from forming free-radicals; supress tumour growth and prevent tumour formation in animal models. Transformation of human lactoferrin gene to tomato may elevate the transgenic tomato's 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 tomato are created. Factors including medium and culture condition are combined together to study the most efficient protocol of tomato tissue culture and plant regeneration thereof. The best recipe for bud differentition is MS (MS with 2mg/L ZT and 0.2 mg/L IAA), the ideal recipe for plant regeneration is MS free of hormone.A plant expression vector pBIhLF, containg marker gene, was constructed. T-DNA cassette of pBIhLF include gene of interesl(hlf gene) driven by CaMV 35S-promoter and marker gene(faw). Meanwhile, another marker-free plant expression vector pl3OOUbihLF was constructed. T-DNA cassette of pl3OOUbihLF only contain gene of interest(/z/f gene) driven by the promoter of Ubiquitin. pl3OOUbihLF andpBIhLF binary vectors were transformed into the expression host, Agrobacterium EHA105 by freezing-thawing in liquid nitrogen respectively.An efficient transformation protocol based on Agrobacterium tumefacien EHA105 was created in the study. Good results were gained when preculturedcotyledons as infectious explants were co-cultivated in medium with low concentration of Acetosyringone (20 u M, for 3 days)and of Agrobacteria (OD6oo=0.8-1.0, for 15 minutes)at the present of 50mg/L kanamycin. The maximum of transformation frequency reached at the 3rd co-cultivation.PCR analysis and Southern Hybridization confirmed that hlf gene had been integrated into genome of tomato.1026 cotyledons of tomato were co-cultivated with Agrobacterium tumefaciens EHAl05/pBIhLF, and 5 resistant buds were obtained with the top transformation frequency of 0.82% and the average transformation frequency of 0.49%. Two resistant plantlets regenerated into full plants in rooting medium.746 cotyledons of tomato were co-cultivated with two Agrobacterium tumefaciensEtiM05(pBlhU!+ p13OOUbihLF), but no anti-bud was obtained. Therefore, we should develop further study on how to acquire marker-free transgenic tomato by two-strain/two-plasmid co-transformation system.