Molecular Cloning Of Vacular H～+-pyrophosphatase And Its Expression In Latificer Of Hevea Brasiliensis (Rubber Tree)

Two types of pyrophosphatase (PPase) are present in plant tissues: soluble one in cytoplasm(inorganic PPase) and insoluble one on the vacuolar membrane(V-PPase). In addition to hydrolyzing PPi, The V-PPase is assumed to function as a H+-pump.The V-PPase on the membrane of lutoids, an organelle in latificer of Hevea brasiliensis (rubber tree), is a crucial enzyme in regulating rubber biosynthesis, it hydrolyzes the pyrophosphate and joints with the V-ATPase to regulate the cell pH to keep the lutoid stable and maintain the optimum condition for the soluble pyrophosphatase in the latex metabolism. In this research, by using degenerate primers, a fragment of V-PPase gene from the latex of Hevea brasiliensis was obtained, which was used to isolate a full length cDNA clone by utilizing the 5' & 3' Rapid Amplification of cDNA Ends(RACE) technique. And the full length cDNA sequence was submitted to GENEBANK ( AY514019). The cDNA length was 2307bp coding for a protein of 769 amino acids with a molecular mass 84.7kDa.Northern blot indicated that the V-PPase mRNAs in latex was kept at almost the same level within 2 days with the control treated with exogenous JA. Thereafter, its transcript levels increased gradually, and reached a peak on the sixth day. The same case, with the treatment of ethylene, the V-PPase gene expression levels increased after 2 days, and reached a peak on the fourth day, and subsequently remained at a high level for at least 4 days, then decrease to the same level as that of control on the tenth day.The activities of V-PPase were also measured in this research. The results showed that, with the treatment of exogenous JA, the activity of V-PPase in latex increased distinctively after 8 hours, and hit the peak on the fourth day, then decreased gradually but still kept the high level compared with the control. While with the treatment of ET, the activities of V-PPase showed a similar pattern, but much more slower than the samples with inducement of exogenous JA.A fragment of V-PPase gene in latex was expressed in PET prokaryotic expression system successfully and got the expressed protein, which established a foundation for the followed-up research, such as making antibody, analysis of histochemistry and so on.