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,CsLAC4、CsLAC6、CsLAC11、CsLAC12、CsLAC13、CsLAC15和CsLAC17全长片段长度分别为1674、1716、1689、1740、1740、1713和1743 bp,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。CsLAC4、CsLAC6、CsLAC11和CsLAC17在甜橙果实贮藏期间呈上调表达,CsLAC12和CsLAC15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。
【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 were1674,1716,1689,1740,1740,1713,1743 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 Lc LAC,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.
作者
龚意辉
吴志蒙
李论
彭淑君
李鹏
陈致印
GONG Yi-hui;WU Zhi-meng;LI Lun;PENG Shu-jun;LI Peng;CHEN Zhi-yin(School of Agriculture and Biotechnology,Hunan University of Humanities,Science and Technology,Loudi,Hunan 417000,China;Xiangtan City Agriculture Scientific Research Institute,Xiangtan,Hunan 4171134,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2021年第12期3195-3204,共10页
Journal of Southern Agriculture
基金
湖南省自然科学基金项目(2020JJ5270)
湖南省科技创新计划项目(2020RC1014)
湖南省教育厅科学研究项目(20A281)。
关键词
柑橘
漆酶
褐斑病
酚类化合物
基因表达
citrus
laccase
peel pitting
phenolic compounds
gene expression Foundation item:Hunan Natural Science Foundation(2020JJ5270)
Science and Technology Innovation Project of Hunan(2020RC1014)
Scientific Research Project of Hunan Provincial Department of Education(20A281)
作者简介
第一作者:龚意辉(1988-),https://orcid.org/0000-0001-5862-0948,博士,主要从事园艺产品保鲜技术研究工作,E-mail:gyhzgh@163.com.主持或作为主要成员参与湖南省自然科学基金项目“漆酶在柑橘褐斑病中的作用机制”、湖南省教育厅科学研究项目“基于宏基因组学探讨EGCG@β-CD纳米粒增强降血脂功效的途径及机制”和“漆酶在黄桃褐变中的作用”等科研项目3项,在《Food Chemistry》《Plant Physiology》《Postharvest Biology and Technology》《Food Chemistry Molecular Science》《International Journal of Molecular Sciences》《南方农业学报》《包装工程》《食品与发酵工业》等国内外期刊发表学术论文18篇,其中第一作者或通讯作者11篇(SCI收录期刊3篇)。;通讯作者:陈致印(1981-),https://orcid.org/0000-0001-7701-7992,博士,副教授,主要从事园艺产品加工研究工作,E-mail:772612626@qq.com。
