Tobacco Transformation Of Thymosin Alpha 1 Gene ByAgrobacterium-mediated Method And Study Of Tissueculture Of Notholirion Bu

Thymosin alpha l(Toti) is a small molecular polypeptide produced from thymic and it is a immunopotentiating agent which is widely used in clinic.Presently, Thymosin alpha 1(T a i) in market was chiefly obtained from chemical product or extraction from calf thymic. Toe i which come from calf thymic is expensive and is limited by material resources. Synthetic thymosin alpha 1 not only pollute the environment but also is expensive. It is a good way to make thymosin alpha 1 by genetic engineering which may resolve above disadvantages.In this study, a plant expression vector pCAMBIA2301 T a \?was constructed . It contained four synthetic repeated Ta i gene under control of CaMV35S promoter between Hindlll and EcoRI sites. The vetcor was transferred into tobacco by Agrobacteerium-mediaied transformation. The aim of the study was to get a transgenic tobacco of expressing thymisin alpha 1 gene and lay a foundation for scale production of T a ] from transgenic plant.The result is following:1. T a i gene was transferred into tobacco by Agrobacteerium-mediated method. A lot of kanamycin resistant regenerated plants were obtained. The transgenic tobacco regeneration rate is 0.31-2.82;2. GUS assay the kanamycin resistant transgenic tobacco by histochemical staining method. The result showed that the GUS positive rate was 0.85%-19.23%. 17 GUS positive plants were got;3. Southern Dot Blot further assay GUS positive tobacco and get 13 Dot Blot positive plants which contained T a 10 gene;4. 13 transgenic tobacco plants,which were southern positive, were successfully transplant to soil and have flowered.The main conclusions were as follows: 1. Toci gene could transferred into tobacco transformation by Agrobacteerium-medialed method. A regeneration system of transgenic tobacco were built. 2. A method of GUS detection was built in transgenic tobacco. 3. Southern Dot Blot confirmed that T a i gene have been intergrated into tobacco genome. The transgenic tobacco of Tai gene were obtainedfor the first time in domestic. 4. A DNA extraction method were built from tobacco.Not only the quantity of DNA is higher but also the DNA is not polluted by RNA.The result will lay a foundation for studing the activity of expressing T a i and producing Thymosin alpha 1 and active polypeptide by transgenic plant.The notholirlon bulbuliferum(lingelsh)stearn is a rarely plant agent. Recently, it were found that notholirion bulbuliferum(lingelsh)stearn own activity to resist cancer. Now it is limited by the resource and can't satisfy the need of the market.The experiment has studied the tissue culture and plant regeneration of notholirion bulbuliferum(lingelsh)stearn .The result showed :(l)bulbule sterilized were cultured on MS basic medium supplemented with different concentration of KT,BA,IAA,NAA and 2,4-D. the calli were induced from bulbulle on the mediums. The induction rate of calli were higher on MS+ NAA 0.5mg/L + KT O.lmg/L medium and grew quickly.(2)The bulbule which were cultured on MS+2.4-D 1.0 mg/K+ KT O.lmg/L medium had bring up new bulbule by adventitious root and the regeneration system of notholirion bulbuliferum(lingelsh)stearn bulbule were built.The experiment confirmed that tissue culture were efficient in keeping resource of notholirion bulbuliferum(lingelsh)stearn species. The study lay a foundation for rapid propagation of notholirion bulbuliferum(lingelsh)stearn and the massive extraction of active substance.