荣霞等

摘 要 以来源于橄榄成熟胚的橄榄试管苗为材料，进行PPO基因克隆，比较试管苗不同保存阶段PPO基因转录水平和PPO酶活性的变化。结果表明：克隆获得1个PPO基因全长cDNA序列，命名为PPO2，GenBank登录号为JQ319005，cDNA全长序列为1 934 bp，推测出橄榄PPO开放阅读框（ORF）1 818 bp，编码606个氨基酸。橄榄PPO2属于碱性、亲水、非分泌蛋白，可能定位于线粒体基质空间，具有3个典型的保守结构域，丝氨酸磷酸化占主导地位，并且二级结构以不规则卷曲为主。而三级结构预测表明，橄榄试管苗PPO2蛋白主要由α螺旋和不规则卷曲组成；进化树分析显示橄榄PPO蛋白与胡杨PPO关系最近，而与葡萄和莲的亲缘关系较远；qPCR分析显示，PPO2基因在橄榄试管苗不同保存阶段都有表达，但在橄榄试管苗保存1个月幼叶比较多时PPO2基因的表达量最高；而橄榄试管苗在分别保存1、2、3、4、5个月时PPO活性变化趋势不明显，当保存时间到6、7、8个月时PPO活性急剧增加。因此，PPO基因可能与幼嫩组织的启动分化有关，并在橄榄试管苗生长发育过程中发挥重要作用。

关键词 橄榄；PPO；克隆； qPCR；酶活性

中图分类号 S667.5 文献标识码 A

Cloning and Expression of Polyphenol Oxidase（PPO）Gene

During Different Process of Preservation in in vitro

Chinese Olive（Canarium album）

RONG Xia， LAI Zhongxiong*， LIN Yuling， LIU Shengcai

LAI Gongti， CHEN Yukun， ZHANG Zihao

Institute of Horticultural Biotechnology， Fujian Agriculture and Forestry University， Fuzhou， Fujian 350002， China

Abstract Chinese olive test-tube plantlets germinated from mature embryo were used as the experiment materials. Cloning and analysis of PPO， the transcriptions of PPO at different preservation stages in in vitro Chinese olive plantlets were studied，and the activity of PPO enzyme was also determined. The results indicated that 1 934 bp full-length cDNA sequence of PPO gene was obtained. It was registered in GenBank， and the accession number was JQ319005. The cDNA contained a 1 818 bp open reading frame（ORF）， encoding 606 amino acids. Besides， a partial sequence of PPO was obtained（PPO1， JF811036）. The predict of the protein showed PPO2 was alkaline， hydrophilic and non-secretory. PPO2 protein contained three typical conserved domains and might localize in mitochondrial matrix space. Serine predominance in phosphorylation， secondary structure was mainly as irregular curl. Three-dimensional structure prediction showed that Chinese olive in vitro plantlets PPO2 protein was surrounded with α-helice and irregular curl. Phylogenetic tree analysis of PPO2 showed there was a close relationship with Populus euphratica， but with a long distance with Vitis vinifera and Nelumbo nucifera. The analysis of qPCR showed preservation for 1 month， the plantlets with more young leaves， and the PPO2 gene expression was at the highest level. PPO activity of Canarium album plantlets changed slightly after preservation for 1～5 months； after preservation for 6～8 months， the PPO activity increased sharply. This research suggested PPO gene might be associated with the initiation of differentiation of tender tissue and organ， and PPO likely played an important role in the process of plantlet growth and development in Canarium album.

Key words Canarium album； Polyphenol oxidase； Cloning； qPCR； Enzymatic activity

doi 10.3969/j.issn.1000-2561.2014.04.021

橄榄（Canarium album L.）属于橄榄科橄榄属，又称青果、白榄、山榄等，是中国南方特有的亚热带果树之一，广泛分布于福建、广东、广西、贵州等省（区）[1]。橄榄不仅果实营养物质丰富，还是重要的药用植物[2]。据《本草纲目》记载，橄榄具有健脾胃、助消化、止泄泻、生津止渴、清热解毒等功效[3]，还具有降压、抗癌、抗菌消炎等作用。橄榄是一种多年生常绿高大乔木，四季常青，树形美观，对绿化、美化环境很有价值。……