A strong promoter and transit peptide sequence are critical components to accumulate a valuable peptide such as antibody or pharmacologically important enzyme in transgenic plants.
ADP-glucose pyrophosphorylase (AGPase; EC 2.7.7.27) is involved in the pivotal step of starch synthesis in plant and strongly induced as sucrose level is increased during starch accumulation in storage organ such as grain or potato tuber. In this study, the promoter and transit peptide sequence of AGPase gene isolated from sweetpotato, ibAGPl was attached to GUS as a reporter gene and transformed into potato in comparison with a constitutive promoter, CaMV35S. The results on the induction activity of GUS enzyme in transgenic potatoes are summarized as follow:
1. Twenty-eight transgenic lines for CaMV35S, 42 for ibAGPl, and 38 for ibAGPl+TP construct were selected against high concentration of kanamycin and PCR analyses and Northern blot hybridization confirmed that recombinant T-DNA constructs are successfully integrated into the potato genome and actively transcribed.
2. Higher level of GUS transcript were detected in CaMV35S lines than those of other constructs (ibAGPl and ibAGPl+TP) and in general, more transcripts were accumulated in leaf tissues than in tuber.
3. In leaf tissues, the GUS activity of CaMV35S line was 1.5-fold greater than that of ibAGPl line but it was dramatically increased (7.5-fold) in ibAGPl十TP line indicating that the transit peptide sequence of ibAGPl was still active in the leaf tissue of heterologous organism, potato.
4. In tuber tissue of ibAGPl line, however, the GUS activity was significantly increased up to approximately 15.6-fold compared with that of CaMV35S line. In addition, the transit peptide in ibAGPl노TP line directed to enhance further up to more than 2-fold than that of ibAGPl line. Ultimately, ibAGPl+TP line elevated the level of GUS activity up to 26.0-fold in tuber compared with that of CaMV35S line. This preliminary results clearly indicated that the expression of ibAGPl is strongly induced as sucrose level is increased in growing tuber of potato.
5. To further investigate the induction of GUS activity by the exogenous sucrose, the transgenic lines were cultured on the MS media without and with 3.0% sucrose. The results showed that high concentration of sucrose induced the GUS activity up to approximately 2.0-fold in both ibAGPl and ibAGPl+TP line. The histochemical analyses exhibited that ibAGPl promoter directed the expression of GUS gene mainly in the actively growing root zones including root cap and active cell division (expension) zones while ibAGPl+TP construct led to be expressed in the entire area of root tissues.
In conclusion, the preliminary results obtained in this study indicate that the ibAGPl promoter and its transit peptide are useful to develop a vector system for the mass production of a useful peptide in tuber tissue of potato.