桃果实铜锌超氧化物歧化酶基因PpCuZnSOD的克隆与分析  

Cloning and Analysis of Copper-zinc-superoxide Dismutase Gene(PpCuZnSOD) from Prunus persica

作　　者：吴军帅[1] 李培环[1] 李鼎立[1] 董晓颖[1] 段艳欣[1] 

机构地区：[1]青岛农业大学园林园艺学院/青岛市现代农业质量与安全工程重点实验室,山东青岛266109

出　　处：《核农学报》2013年第4期408-417,共10页Journal of Nuclear Agricultural Sciences

基　　金：国家青年基金(NSFC;30900976)

摘　　要：从已构建的硬肉桃(Prunus persica L.)双久红果实SSH文库中获得4条铜锌超氧化物歧化酶(CuZnSOD)基因的ESTs,通过blast比对和电子克隆后进行了序列拼接和经RT-PCR方法,获得桃果实铜锌超氧化物歧化酶基因PpCuZnSOD全长cDNA序列(GenBank登录号:JX217743)。该基因全长665bp,包含一个459bp的ORF(120bp-578bp),编码152个氨基酸,推测蛋白分子量为15.38kDa,理论等电点为5.60。该基因具有典型的高度保守的Cu2+和Zn2+活化中心及特征信号,其氨基酸序列与其他植物的CuZnSOD具有很高的相似性。生物信息学分析结果表明,该蛋白定位于细胞质中,是非跨膜的非分泌性亲水蛋白。二级结构分析结果表明,该蛋白主要由无规则卷曲和延伸链等蛋白质二级结构元件构成,三维结构预测结果显示,PpCuZnSOD有3个转角环和8个折叠构成桶状的活性中心。系统进化分析结果表明PpCuZnSOD与小金海棠(Malus xiaojinensis)sod2同源关系最近,而与荔枝(Lichichinensis)、龙眼(Dimocarpus longan)和拟南芥(Arabidopsis trifoliate)等亲缘关系较远。QPCR结果表明,PpCuZnSOD在幼果中表达量最高,雌蕊和雄蕊次之,花瓣和叶片中表达量最少;果实成熟前后,PpCuZnSOD在硬肉芽变桃双久红果实中的表达量显著高于软肉桃川中岛白桃中的表达量,表明该基因可能在维持果实质地和硬度方面有一定作用。本研究结果为进一步研究该基因在桃果实成熟软化中的作用奠定了基础。Four copper/zinc-superoxide dismutase gene ESTs from constructed SSH library of stony hard peach （Prunus persica L. ） ＇ Shuangjiuhong＇ were found, and after sequence blasting, silico cloning, sequence splicing, and RT-PCR, a full-length eDNA sequence of P. persica copper-zinc-superoxide dismutase gene （PpCuZnSOD, GenBank Accession no： JX217743） was isolated. The full-length eDNA of PpCuZnSOD was 665bp, including a 459bp open reading frame （ORF） which encodes a 152-amino-acid protein. The predicted molecular weight of the protein was 15.38 kDa with the pI of 5.60. PpCuZnSOD contained typical highly conserved enzyme core of Cu2 ＋ and Zn2 ~ , and was highly similar to others. Bioinformatic analysis results showed that the protein was located in the cytoplasm and was a hydrophilic, non- transmembrane and non-secretory protein. The main motif of PpCuZnSOD in secondary structure was random coils and extended strands. The three-dimension structure modeling showed that PpCuZnSOD contained 3 helixes and 8 sheets, and had a cylinder motif and three external loops in the enzyme core. The phylogenetic tree suggested that PpCuZnSOD was closest to Malus xiaojinensis sod2 and farther to Lichi chinensis, Dimocarpus longan, and Arabidopsis trifoliate. QPCR revealed that the expression of PpCuZnSOD was the highest in young fruits, followed by pistils and stamens, and least expression was observed in leaves and petals. Before and after fruit ripening, the accumulation of PpCuZnSOD was significantly higher in the firm-fleshed mutant ＇ Shuangjiuhong＇ than that in the soft-fleshed ＇ Kawanakajima Hakuto＇ , which indicated that PpCuZnSOD might play a role in maintaining the fruit texture and firmness. This study laid the foundation for further research on the role of PpCuZnSOD in peach fruit ripening and softening.

关 键 词：桃 PpCuZnSOD基因 克隆 生物信息学 基因表达 

分 类 号：S662.1[农业科学—果树学]