芒果果实多酚氧化酶(PPO)基因的克隆与表达分析

Cloning and Expression Analysis of Polyphenol Oxidase(PPO)Gene from Mango Fruit

多酚氧化酶(Polyphenol oxidase,PPO)是引起水果褐变的关键酶。为了研究芒果果实中PPO基因的功能,本试验采用RACE方法从‘贵妃’芒果果实中克隆得到了1个PPO基因,该基因cDNA序列全长为1930bp,开放阅读框为1782bp,编码593个氨基酸。芒果PPO基因编码的多酚氧化酶属于亲水性蛋白质,分子重量为66.82KD,等电点为6.95,不含跨膜结构和信号肽,为非分泌蛋白,包含Tyrosinase、PP01_DWL和PP01_KFDV保守结构域。芒果PPO蛋白的二级结构无规则卷曲、β-折叠和α-螺旋占比分别为63.58%、22.60%和13.83%,三级结构与模板6els.1.A-致性达70.94%,可能与C2H2 and C2HC zinc fingers superfamily protein(TT1),Multidrug resistance protein,mate family(TT12)和Autoinhibited H(+)-ATPase isoform 10(AHA10)等存在相互作用。聚类分析表明该蛋白与开心果、橄榄、克莱门柚和甜橙等植物的亲缘关系较近。通过qRT-PCR分析,芒果PPO基因主要在‘桂七’果皮中表达量较高,而在‘贵妃’果皮中表达量较低。该基因的克隆及其功能的研究对芒果抗酶促褐变品种的培育以及分子改良具有重大意义。

Polyphenol oxidase(PPO)is a key enzyme that causes fruit browning.In order to study the function of PPO gene in mango fruit,a PPO gene was cloned from the'Guifei'mango fruit by RACE method.The full-length cDNA sequence of PPO gene was 1930bp and the open reading frame was 1782bp,encoding 593 amino acids.Polyphenol oxidase encoded by PPO gene of mango belonged to hydrophilic protein with molecular weight of 66.82KD and isoelectric point of 6.95.It was a non-secretory protein without transmembrane structure or signal peptide,containing the conserved domain of Tyrosinase,PPO1_DWL and PPO1_KFDV.The proportions of random coil,β-pleated sheet and a-helix in the secondary junction were 63.58%,22.60%and 13.83%.The consistency of the tertiary structure with the template 6els.1.A was 70.94%.There might be interactions with proteins such as C2H2 and C2HC zinc fingers superfamily protein(TT1),Multidrug resistance protein,mate family(TT12),and Autoinhibited H(+)-ATPase isoform 10(AHA10).Cluster analysis showed that PPO protein of mango was closely related to pistachio,olive,citrus clementina and sweet orange.By qRT-PCR analysis,PPO expression level was higher in'Guiqi'pericarp,but lower in'Guifei'pericarp.The cloning of this gene and the study of its function were of great significance for the breeding and molecular improvement of mango resistant varieties to enzymatic browning.