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

【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 CsLAC4，CsLAC6，CsLAC11，CsLAC12，CsLAC13，CsLAC15，CsLAC17 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 CsLAC4，CsLAC6，CsLAC11，CsLAC17 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.