Design, Cloning And Construction Of The Human Proinsulin Gene, And Its Transformation To Potato

Diabetes mellitus is the third highest disability and death rate disease following cardiovascular diseases and cancers.It is a great global task by the WHO to combat the disease in order to protect human health.At present,insulin is admitted that it is the most effective drug for diabetes treatment.However, insulin can not be used widely nowadays because its production cost is still too high for many patients to use it lifelong although it could be produced from genetic engineered bacteria or yeast.It could be solved this problem to utilize transgenic plants to produce insulin.In this paper,a plant expression vector containing human insulin gene has been constructed and then transformed into potato (Solanum tuberosum cv. Atlantic) through Agrobacterium-mediated transformation. This achievement made important progress to produce cheaper human insulin in genetic engineered potato.Results of this paper were abstracted as follows:1,A promoter of PatatinⅠ,a tuber-specific-promoter,was amplified, cloned and sequenced from Solanum tuberosum cv.Long 3(GenBank:GU168944).The cloned fragment is 1100bp long and contains the key cis-elements, such as TATA box, CAAT box, B-box and transcription start region. It shared 96% homology with a reported sequence in GeneBank(CQ876983).2,According to whether it contains signal peptide gene, two primers were designed and synthesized respectively, which had NcoⅠrestriction enzyme site. Tuber-specific-promoter Patatin was sub-cloned and ligased with pMD18-T plasmid. Then recombinant plasmid pGPP05 (without singal peptide gene) and pGPP06 (without singal peptide gene) were obtained.3,According to the potato bias codons and the amino acid sequence of human proinsulin from GeneBank, Human proinsulin gene was designed and synthesized with NcoⅠrestriction enzyme site in the front of human proinsulin sequence and stop codon and BstEⅡrestriction enzyme site in the end of human proinsulin sequence. Then, recombinant plasmid pMD-hINS was obtained by ligation of pMD18-T plasmid and the human proinsulin gene.4,Plant expression vector p1301P05h(without singal peptite gene) and p1301 P06h (within singal peptite gene) which contain patatin promoter and human proinsulin gene were constructed.Then they were trandformed into Agrobacterium strain LBA4404 respectively by freeze-thaw method.It was evidenced that vectors had been transferred into LBA4404.5,Regeneration medium of 4 potato varieties,Atlantic,Favorita,Shepody and GanNongshu NO.2 was screening.The optimal medium for callus induction from stem was MS+2.0mg/L6-BA+0.2mg/LNAA for Atlantic,MS+2.0mg/L6-BA+0.5mg/L NAA for favorite,MS+2.5mg/L6-BA+0.2mg/LNAA for Shepody and GanNongshu NO.2;The optimal medium for adventitious bud differentiation from stem-derived calluses was found to be MS+2.5 mg/L 6-BA+5.0mg/LGA3 for Atlantic and GanNongshu NO.2,MS+3.5mg/L 6-BA+10mg/L GA3 for Shepody, MS+2.5mg/L 6-BA+0.1mg/L NAA+10mg/L GA3 for Favorita.6,The optimum conditions of agrobacterium-mediated transformations was established.Atlantic stem explants were pro-cultured for 2 days,infected with OD600=0.5 LBA4404 for 8～10min, co-cultivated for 2 days on culture medium. Explants needed different selective concentration of hygromycin at different culture stage.The concentration was 25mg/L at the stage of callus and shoot formation.The concentration was 15mg/L at the stage of root formation.7,By the established LBA4404-mediated genetic transformation system of Atlantic, we obtained Hgy resistant seedling in rooting medium.