Cloning And Expression Of A Tonoplast H~+-PPase Gene From Cucumis Sativus L.

Tonoplast H~+-PPase is a common proton pump in plants, and it plays a vital role in maintaining ionic equilibrium in cells; it also controlls the tranportation of phytohormone as a regulatory factor and so forth plays a role in regulating the growth and development of plants. Cucumber is one of the main vegetables in China. In the cultivation of it, salt, freezing, drought and other stresses are often encountered. Resistance of plants to several stresses is closely related with tonoplast H~+-PPase. Recently, with the increasing cultivation area of cucumber, salt and temperature stresses have become a limited factor for cucumber production, therefore, its meaningful to increase cucumber tolerance to stress through some method including gene utilization. In this study, the full-length cDNA of tonoplast H~+-PPase was amplified by RT-PCR and RACE methods from young leaves of cucumber (Cucumis sativus L.). Furthermore, we constructed the sense and the antisense expression vectors. Tomato transgene was studied using Agrobacterium-mediated plant transformation. The gene expression of tonoplast H~+-PPase under several stresses was also studied using RT-PCR methods. The main results were as follows:1. Homologous sequence design were applied to annex primer. With RT-PCR and RACE methods, a cDNA of tonoplast H~+-PPase, named CuPPase, was amplified from cucumber (Cucumis sativus L.). It's accession number in GenBank was GQ223786.2. Sequencing analysis showed that the full cDNA length of CuPPase consisted of 2,650 bp which contains an open reading frame of 2,307 nucleotides coding for a 768-amino acid protein of 80 kD. The predicted PI was 5.32. 14 transmembrane helices were found in the gene and the result of PlantCare analysis showed that it had the property induced by abscisic acid, auxin, gibberellin, salicylic acid, freezing, drought and other factors. 3. RT-PCR showed that CuPPase had high expression in leaf and root while had low expression in stem. Expression of CuPPase was induced by salt, high and low temperature stresses, and the induction level was related with treatment time. 4. In order to determine the function of tonoplast H~+-PPase in cucumber, a sense and an antisese vector were constructed for plant transformation.