Transformation Of Thymosin Gene Into Lettuce And Selection And Detection Of Transgenic Lettuce

Thymosin (THY) is peptide hormone that secrete from human's or animal's thymus tissue. The thymosin is important in maturation, differentiation and immunity of lymphoid leucocyte. Especially, the thymosin plays a vital role in resistance to tumor, transplantation and infection of microbion. The thymosin being used in clinic is separated and purified from thymus of animals, but the thymosin produced in this way is expensive and too little. Although the thymosin has been successfully expressed in the prokaryotic cell system, its application is restricted because of its deficiency of modification after translation. Therefore, the transgenic plant, which used as a bio-reactor ,will produce more thymosin to meet the increasing demand of thymosin.The aim of this study was to transform the thymosin gene into lettuce mediated by Agrobacterium tumafaciens and to select transgenic lettuce and confirm them. The main results are as following:1. The effects of different hormone concentration on shoot regeneration of lettuce cotyledon explants were investigated, and the results showed that the suitable medium for shoot regeneration is MS+0.1mg/L 6-BA+0.05mg/L NAA and the shoot regeneration efficiency is 60.3%; The experiment about sensitivity of cotyledon to kanamysin and carbenicillin showed that the suitable kanamysin concentration for selecting transgenic tissue is 75mg/L and the carbenicillin concentration is 500mg/L.2. The thymosin gene was transformed into lettuce cotyledon explants mediated by Agrobacterium tumafaciens. The explants were planted on MS+0.1mg/L 6-BA+0.05mg/LNAA+75mg/Lkan+500mg/LCarb and root was induced on l/2MS+50mg/LKan+100mg/LCarb. 256 kanamycin-resistant plants were obtained. PCR detection showed that 12 plants were positive and the positive frequency was 4.6%.3. Southern blot analysis of PCR positive plants indicated that 3 plants weretransgenic plants and the thymosin gene has been transformed into genome of lettuce.4. RNA of transgenic lettuce was extracted and RT-PCR detection preliminarily proved that the thymosin gene was expressed in transcriptional level.