关键词: 黄瓜, cDNA 克隆, 序列分析, 表达

Abstract: Using PCR degenerate primers designed based on the conserved amino acid sequences of known tonoplast H⁺-PPase to amplify cDNA fragments from cucumber by RT-PCR and RACE-PCR, a H⁺-PPase gene named CuPPase was cloned. The CuPPase protein showed 94%, 92% similarity to the H⁺-PPases from Cucurbita moschata and Vigna radiata. Bioinformatics analysis indicated that the full-length cDNA sequence was 2 650 bp, which contained an open reading frame of 2 307 bp and encoded a protein of 768 amino acid residues with a calculated molecular weight of 80 kD and isoelectric point of 5.32. There was fourteen strong inside to outside transmembrane helix. PlantCare analysis indicated that there were abscisic acid, somatotropic hormone, salicylic acid cis-acting responsive elements and gibberellin and low temperature，drought wound-responsive elements in the protein encoded by CuPPase. The result of RT-PCR analysis indicated that CuPPase expressions were different in roots，stems and leaves，and richer in leaves and roots, poorer in stems. The expression of CuPPase is induced by salt stress and low or high temperature stress, but is closely related to the processing time. Its expression trend increased firstly and then decreased.

Key words: cucumber, cDNA cloning, sequence analysis, expression