Transformation Of Human PIns And GUS Gene In Potato And Arabidopsis Thaliana Under The Control Of Class ? Patatin Promoter

To produce pharmaceutical proteins,plant bioreactors are low cost.Potato tuber is such a bioreactor with a high yield.Transgenic potato with a high-value-protein encoding gene could increase potato industry output.For example,we can get not only starch products but also polypeptide drugs such as insulin from an insulin-encoding gene transferred potato.As a symbolic protein in potato tuber,patatin contributes up to 40%of the total soluble proteins in potato tubers.Expression of Class?patatin-encoding genes is tuber-specific and sucrose-inducible.It is not clear whether the promoter has any other responsive characteristics and whether the 23 amino acid residues of a patatin amino terminal can play a signal role.The main results in this paper are as follows:1.Induction of microtubers.Compared with explants induced by the F1 and F2 metheds,the F3 methed is better to induce microtubers.Days for microtuberization were shortened by about 12 days if explants are cultivated by the F3 methed.By this methed,a ratio of tuberizing explants achieved 86.15%and a ratio of big-tuber explants is about 32.35%when explants were induced by the inducing medium D.2.In total,61 transgenic potato plants with Pro_Patatin::GUS?StG2G series?were constructed,42 plants with Pro_Patatin::Signal-GUS?StG2SG series?,45 plants with Pro_Patatin::PIns?StG2I series?,and 52 plants with Pro_Patatin::Signal-PIns?StG2SI series?.All of these transgenic plants were confirmed by PCR with a pair of gene-specific primers.Meanwhile,they were all verified by PCR with a pair of agrobacterium-specific primers.3.Microtubers of St G2G series and St G2SG series plants were histochemically assayed for GUS.Parts of the microtuber cortexes were stained a light blue color.The other parts were not stained.The whole explants of the StG2G series and StG2SG series were not stained.Accumulation of human proinsulin in roots,stems and microtubers of the StG2I series and StG2SI series transgenic plants can not be detected by Western Blotting assay.4.AtSG series transgenic Arabidopsis thaliana plants were constructed with the class I patatin promoter driven Signal_Patatin-GUS fusion genes in pCAMBIA1301.After histochemically stained,cotyledons and roots of the transgenic plants became blue,but not hypocotyl.In the cotyledons,vascular bundles and wooly hairs were darker blue.5.The transgenic Arabidopsis plants?AtG series and AtSG series?responded to drought,cold,darkness and other environmental signals,and the most sensitively to cold.6.Moreover,these transgenic plants?At G series and AtSG series?responded to auxin?IAA,NAA?,cytokinin?6-BA,ZT?,gibberellin?GA₃?,ABA.Compared with NAA,the promoter is more sensitive to IAA.The promoter is induced by ZT than 6-BA.Response of the promoter to ABA is weak.GA₃ is a little lower than NAA.7.The patatin promoter of the transgenic Arabidopsis plants?AtG series and At SG series?was also induced by three disaccharides?sucrose,lactose,maltose?and two monosaccharides?fructose and glucose?.The induction by the disaccharides was significantly stronger than by the monosaccharides.The induction by sucrose was concentration-dependent.The promoter can be induced by as low as 10 mM of sucrose.The induced GUS activity of transgenic Arabidopsis plants by 300 mM was ten times higher than 10 mM and five times higher than100m M.8.The signal peptide,23 amino acid residues of the patatin amino-terminal has a significant effect on GUS activity.The activity of the Signal_Patatin-GUS fusion enzyme is about four times higher than that of only the GUS.