关键词: 豆梨, 植物络合素合酶, 基因, 克隆, 丁胱亚磺酰亚胺, 表达特点

Abstract: Pear rootstocks bean pear（Pyrus calleryana Dcne.）was used as an experimental materials for cloning phytochelatin synthase gene and researching its expression characterization via rapid amplification of cDNA ends（RACE）and fluorescent quantitative reverse transcription polymerase chain reaction（RT-PCR）methods. PcPCS1 cDNA sequence length is 1 970 bp, which contains an open reading frame that would encode a 54.77 kD protein of 497 amino acids. Two typical phytochelatin subfamily domains constitute PcPCS1 protein, which includes three adjacent Cys-Cys elements（331-332, 351-352 and 369-370 location amino acid）in the C-terminal region and all characteristics sites（Cys-56, Cys-90/91 and Cys-109）of phytochelatin synthase protein. PcPCS1 and other PCS1 proteins from leguminous plants belong to the same branch in PCS1 phylogenetic tree. Moreover, PcPCS1 had the highest homology（84%）with Lotus corniculatus LjPCS1. Fluorescence quantitative RT-PCR results showed that PcPCS1 gene was constitutively expression and its expressions were different in various organs. Furthermore, PcPCS1 transcription level was quickly climbed up after 20 mmol · L-1 CdSO4 treatment 24 hours, which was higher in leaf than in root. It was very significant that three heavy-metal ions had different abilities to up-regulated PcPCS1 expression as the sequence Cd2+ > Zn2+ > Cu2+. It is need to point out that PcPCS1 expression was significantly inhibited by 200 mmol · L-1 L-Buthionine sulfoximine（BSO） which was one of γ-glutamyl cysteine synthetase inhibitors. However, its expression returned to the normal level of single heavy-metal treatment or exceeded it after adding 200 mmol · L-1 L-Glutathionereduced（GSH）to the nutrient solution. In summary, the bean pear phytochelatins synthesis used glutathione as substrate, which was produced through γ-glutamyl cysteine synthetase pathway.

Key words: Pyrus calleryana Dcne., phytochelatin synthase, gene, cloning, L-Buthionine sulfoximine, expression characteristics