豆梨植物络合素合酶PcPCS1基因克隆及其表达分析  

Molecular Cloning and Expression Analysis of a Phytochelatin Synthase Gene,PcPCS1,from Pyrus calleryana Dcne.

作　　者：李慧[1,2] 丛郁[3] 王宏伟[1] 盛宝龙[1] 蔺经[1] 常有宏[1,2] 

机构地区：[1]江苏省农业科学院园艺研究所,南京210014 [2]江苏省食品质量安全重点实验室,南京210014 [3]中国科学院南京土壤研究所,南京210009

出　　处：《园艺学报》2010年第6期880-890,共11页Acta Horticulturae Sinica

基　　金：江苏省农业科学院科研基金项目(6110816);江苏省科技基础设施建设计划项目(BM2008008);国家农业科技成果转化资金项目(2008GB2C100100)

摘　　要：以梨砧木豆梨(Pyrus calleryana Dcne.)为试材,克隆其植物络合素合酶基因(PcPCS1)的cDNA全长序列,并研究该基因的表达特点。结果表明PcPCS1 cDNA序列长度为1970bp,编码497个氨基酸,推导的氨基酸序列由两个典型的植物络合素亚家族结构域组成,具有3个相邻的Cys-Cys元件(331-332、351-352和369-370位氨基酸)和植物络合素合酶蛋白的特征位点(Cys-56、Cys-90/91和Cys-109)。它与豆科植物的PCS1蛋白处于系统发生树的同一分支,且与百脉根LjPCS1蛋白的同源性最高(84%)。荧光定量RT-PCR分析结果显示:PcPCS1基因为组成型表达,各器官表达量存在差异,在20μmol·L-1CdSO4胁迫条件下,其表达量迅速上升,并且在豆梨叶中的表达量高于根;不同重金属离子对其上调表达的诱导能力为Cd2+>Zn2+>Cu2+。200μmol·L-1γ–谷氨酰半胱氨酸合成酶抑制剂丁胱亚磺酰亚胺能够显著抑制该基因的表达,补充200μmol·L-1谷胱甘肽后,其相对表达量恢复或超过单一重金属离子处理的表达水平,即豆梨植物络合素以谷胱甘肽为底物,通过γ–谷氨酰半胱氨酸合成酶途经合成。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 μmol · 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 μmol · 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 μmol · 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.

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 

分 类 号：S661[农业科学—果树学]