Recombinant Sct-cgrp Gene The Lettuce System To Establish And Expression

Cardiovascular disease and Osteoporosis do great harm to the mankind's health, especially impact on the aged persons'life. Nowadays, curing two diseases mainly use salmon calcitonin(sCT) and humanαcalcitonin gene-related peptide(hαCGRP) respectively. But clinical sCT and hαCGRP are attained by tissue extract or chemical synthesis, the method not only was high-cost but also limited sources.In order to prevent and treat cardiovascular disease and osteoporosis at the same time, this experiment was carried out to study the recombination of msCT and hαCGRP fusion gene. The fusion gene has been cloned into the plant binary Ti vecter system. We plan on expressing this fusion gene by lettuce, pepole responds well to treatment through eat transgenic lettuce or extract product.The species of lettuce(Lactuca sativa var.capatata L) were selected and tissue culture system was optimezed. As a result, we drew following conclusions: seeds were sterilized with 8% sodium hypochlorite for 30 min and subsequently sowed on 1/2 MS medium, cotyledon petiole were prepared by cutting of 7-day-old seedling and transferred to the regeneration culture medium by the method of right-side up.Making use of the laboratory stores of Agrobacterium tumefaciens EHA105 harboring the binary vecter pBI AMV/hαCGRP/msCT, agrobacterium-mediated genetic transformation system was optimezed. Finally, we attained following conclusions: Process of transformation contains preculture for 2 days, the concentration of A. tumefaciens suspension OD600=0.1 and infection time 5 min. Selection culture medium supplemented with 500mg/L cefotaxime sodium, 25 mg/L kanamycin on the basis of regeneration culture medium.Ten independent plants of transgenic and resistant lettuce were chosen and reproducted. PCR analysed offspring generations, the work presented here demonstrates that eight transgenic lettuce lines were positive, transgenic positive rate reached 80%. A positive and transgenic lettuce line was chosen for DNA sequencing , the result showed that msCT-hαCGRP fusion gene was successfully integrated into lettuce genome. The new lines lettuce has the function of health care and medical treatment, people responds well to treat through eating it or extracting medical products. The results may lay a solid foundation for studying and development of the medicine.