米良1号猕猴桃漆酶基因的克隆与表达分析  

Cloning and Expression Analysis of the Laccase Genes from Kiwifruit(Actinidia chinensis var. deliciosa 'Miliang-1')

作　　者：高敏霞[1] 冯新[1] 赖瑞联[1] 胡艺贤 陈文光[1] 吴如健[1] 陈义挺[1] 

机构地区：[1]福建省农业科学院果树研究所,福州350013

出　　处：《农业生物技术学报》2018年第1期64-76,共13页Journal of Agricultural Biotechnology

基　　金：福建省自然基金(No.2017J01044);福建省农业科学院博士科研启动基金(No.DC2017-2);福建省农业科学院专项(No.A2016-2);福建省属公益类科研院所科研专项(No.2015R1014-3)

摘　　要：漆酶(laccases)为含铜的糖蛋白,通过保守结构域上铜离子的氧化还原催化各种各样的底物,进而参与植物的各种生理生化进程,如木质素的合成、铁代谢、类黄酮的合成、伤口愈合和抗逆响应等过程。但在果实采后贮藏中的作用未见报道。为了研究漆酶基因在猕猴桃(Actinidia chinensis var.deliciosa)果实贮藏过程中的功能,本研究采用反转录PCR(reverse transcription PCR,RT-PCR)和RACE-PCR法从米良1号猕猴桃(Actinidia chinensis var.deliciosa'Miliang-1')中克隆到2条漆酶基因,分别命名为AdLAC3和AdLAC7。AdLAC3全长序列为2 594 bp,开放阅读框为2205 bp,编码734个氨基酸,登录号为MF405444;AdLAC7全长序列为2 126 bp,开放阅读框为1 695 bp,编码564个氨基酸,登录号为MF405447。此外,还获得了AdLAC3基因的2条可变剪接转录本(AdLAC3-variant1和AdLAC3-variant2),其中AdLAC3-variant1的登录号为MF405445,AdLAC3-variant2的登录号为MF405446。结构域分析显示,AdLAC3和AdLAC7蛋白均具有植物漆酶的3个典型铜离子结合域(Cu-oxidase_3,Cu-oxidase和Cu-oxidase_2),但在系统进化分析中,两种蛋白聚在不同的进化分支,序列差异较大,可能源自不同的基因祖先。AdLAC3和AdLAC7基因均由5个内含子和6个外显子组成。不同组织部位的定量表达分析表明,AdLAC3在根部的表达量最高,其次为茎和叶中,在果实中基本不表达。类似地,AdLAC7基因也在根中的表达量最高,在茎中的表达量次之,在叶和果实中基本不表达。对果实在不同贮藏条件下的qRT-PCR分析结果表明,AdLAC3基因在25℃、4℃和ABA处理的果实中都被显著诱导表达,而AdLAC7基因仅在ABA处理的果实中显著上调表达,说明AdLAC3和AdLAC7基因在猕猴桃果实贮藏中发挥的具体功能不同。本研究为猕猴桃的采后贮藏和品质调控研究提供了新依据。Laccases are multi-copper containing glycoproteins. By catalyzation of various kinds of substrates through the oxidation-reduction reaction of copper ions in their conserved domains, Laccases involve in various physiological and biochemical processes, such as lignin synthesization, iron metabolism, flavonoid biosynthesis, wound healing, stress responses and et al. However, their functions in the post-harvest storage have not been reported. In order to study the function of the laccase genes in the kiwifruit storage process, two laccase genes （AdLAC3 and AdLAC7） were cloned from the Actinidia deliciosa cv. Miliang-1 using reverse transcription PCR（RT-PCR） and RACE-PCR methods. The full-length sequences of AdLAC3 were 2594 bp. It contained a 2205-bp open reading frame, encoding a 734 aa polypeptide and its accession number was MF405444. The full-length sequences of AdLAC7 were 2126 bp. It contained a 1695-bp open reading frame, encoding a 564 aa polypeptide and its accession number was MF405447. In addition, two alternative spliced variants （AdLAC3-variant1 and AdLAC3-variant2） of AdLAC3 were obtained. The accession number of AdLAC3-variant1 was MF405445, and the accession number of AdLAC3-variant2 was MF405446. Protein domain analysis showed that AdLAC3 and AdLAC7 both contained three typical copper binding domains （Cu-oxidase_3, Cu-oxidase and Cu-oxidase_2）, but AdLAC3 and AdLAC7 were distributed to different evolutionary branching in the phylogenetic tree, which was due to the great difference in their sequences and implied that they may originate from different ancestral genes. Both AdLAC3 and AdLAC7 harbored five introns and six exons. qRT-PCR analysis in different tissues showed that AdLAC3 exhibited the highest expression levels in roots, followed by stems and leaves, but almost no expression level was detected in fruits. Similarly, AdLAC7 gene also showed the maximum expression in roots, followed by stems, while almost no expression was observed in the leaves and fruits. qRT-PCR analysis in

关 键 词：猕猴桃 漆酶基因 不同贮藏条件 表达分析 

分 类 号：S663.4[农业科学—果树学]