漆酶基因在柑橘褐斑病发生过程中的表达模式分析

Expression analysis of the laccase gene during the development of peel pitting in citrus fruit

  • 摘要: 【目的】克隆柑橘漆酶基因(CsLAC)并分析其表达规律,为进一步完善柑橘褐斑病的发生机制提供理论依据。【方法】以甜橙为试验材料,利用液相色谱—质谱联用(LC-MS)技术分析柑橘褐斑病发生过程中主要酚类化合物的变化,并采用实时荧光定量PCR分析漆酶基因在柑橘褐斑病发生过程中的表达规律。【结果】PE包装明显降低甜橙果实褐斑指数,利用LC-MS技术在甜橙果皮中共检测到1191个差异离子,共鉴定出18种酚类物质,其中没食子酸(Gallic acid)、儿茶素(Catechin)、表没食子儿茶素(Epigallocatechin)和原花青素B2(Procyanidin B2)的含量在果实贮藏期间呈下降趋势。成功克隆出7个柑橘漆酶基因,CsLAC4CsLAC6CsLAC11、CsLAC12CsLAC13CsLAC15CsLAC17全长片段长度分别为1674、1716、1689、1740、1740、1713和1743bp,分别编码557、571、562、579、579、570和580个氨基酸残基,与参考序列的相似性分别为99.88%、99.83%、99.70%、99.71%、99.60%、99.59%和99.89%。系统发育进化树显示,CsLAC15与荔枝LcLAC的氨基酸序列相似性较高(64.7%),CsLAC12与苹果MdLAC7的氨基酸序列相似性较高(65.7%)。氨基酸保守序列结果表明柑橘漆酶氨基酸序列含有漆酶的3个典型铜离子结构域,分别为Cu-oxidase-3、Cu-oxidase-1和Cu-oxidase-2。CsLAC4CsLAC6CsLAC11CsLAC17在甜橙果实贮藏期间呈上调表达,CsLAC12CsLAC15在果实贮藏期间总体上呈下调表达。【结论】结合甜橙果皮褐斑与CsLAC表达量的关系,推测CsLAC6的大量上调表达可能对柑橘果皮褐斑形成起促进作用。

     

    Abstract: 【Objective】To investigate the expression characteristics of laccase gene(CsLAC)of citrus fruit,to provide a theoretical basis for the mechanism of peel pitting in citrus fruit.【Method】Sweet orange fruit were used as experimental materials. Liquid chromatography-mass spectrometry(LC-MS)was used to detect and analysis the main phenolic compounds during the development of peel pitting at the storage stage,and the expression of laccase genes were monitored during the development of peel pitting in citrus fruit using real-time fluorescence quantitative PCR.【Result】The results showed that peel pitting was inhibited by PE packaging treatment. A total of 1191 differential ions were detected in the peel of sweet oranges by LC-MS and 18 phenolic compounds were identified. The contents of gallic acid,catechin,epigallocatechin and procyanidin B2 were decreased during the storage of citrus fruit. Seven laccase genes were cloned by RTPCR. The full lengths cDNA of CsLAC4CsLAC6CsLAC11CsLAC12CsLAC13CsLAC15CsLAC17 genes were 1674,1716,1689,1740,1740,1713,1743 bp,encoding 557,571,562,579,579,570,580 amino acids residues, with similarities to the reference sequence of 99.88%,99.83%,99.70%,99.71%,99.60%,99.59% and 99.89%,respectively. Phylogenetic analysis showed that CsLAC15 had 64.7% amino acid sequence similarity to litchi LcLAC,while CsLAC12 had 65.7% similarity to apple MdLAC7 amino acid sequence. The laccase amino acid sequences in different plants had a structure of three highly conserved domains:Cu-oxidase-3,Cu-oxidase-1 and Cu-oxidase-2. The expressions of CsLAC4CsLAC6CsLAC11CsLAC17 were up-regulated and CsLAC12 and CsLAC15 were down-regulated during the storage of sweet orange fruits.【Conclusion】Combining the relationship of peel pitting and CsLAC expression,it is suggested that the CsLAC6 in pericarp may contribute to the peel pitting of sweet orange fruit.

     

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