菠萝黑心病发生过程中AcAPXs的表达分析及克隆  

Expression Analysis and Cloning of AcAPXs During the Development of Internal Browning in Pineapple

作　　者：张媛媛[1,2,3] 鹿志伟 李茂富 姚全胜[2] 侯晓婉[2,3] ZHANG Yuanyuan;LU Zhiwei;LI Maofu;YAO Quansheng;HOU Xiaowan(College of Horticulture,Hainan University,Haikou,Hainan 570228,China;South Subtropical Crops Research Institute,Chi-nese Academy of Tropical Agricultural Sciences/Key Laboratory of Hainan Province for Postharvest Physiology and Technology of Tropical Horticultural Products,Zhanjiang,Guangdong 524091,China;Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]海南大学园艺学院,海南海口570228 [2]中国热带农业科学院南亚热带作物研究所/海南省热带园艺产品采后生理与保鲜重点实验室,广东湛江524091 [3]中国农业科学院生物技术研究所,北京100081

出　　处：《热带作物学报》2023年第3期597-606,共10页Chinese Journal of Tropical Crops

基　　金：海南省自然科学基金面上项目(No.320MS089)。

摘　　要：菠萝黑心病(internal browning,IB)严重制约菠萝产业发展,该病害的发生主要是过量活性氧(reactive oxygen species,ROS)引起膜损伤,打破质体多酚氧化酶(polyphenol oxidase,PPO)与液泡内酚类底物原有的区室化划分,使其接触氧化后聚合成醌类物质。抗氧化清除系统能够清除过量ROS,维持机体内ROS的动态平衡。本课题组前期用抗氧化剂抗坏血酸(ascorbic acid,AsA)处理采后‘巴厘’菠萝,能有效延缓黑心病的恶化,然而其作用机理尚不明确。抗坏血酸过氧化物酶(ascorbate peroxidase,APX,EC1.1.11.1)是I类血红素过氧化物酶,以抗坏血酸作为特定的电子供体,催化过氧化氢转化为水,参与植物的多种发育生理过程和胁迫反应。本研究筛选出6个AcAPX基因,对其黑心病发生过程以及AsA处理后的转录水平进行分析,发现AcAPX1基因在贮藏后期极显著响应AsA处理,9 d和12 d时显著上调表达;相关性分析发现,AsA处理后的菠萝黑心病指数与AcAPX1基因表达水平呈显著正相关。结果表明:AcAPX1显著响应AsA处理,可能在AsA延缓菠萝黑心病中发挥重要作用。为了进一步探究其作用机制,从菠萝果肉中克隆到AcAPX1基因,通过生物信息学分析方法对其理化性质、蛋白结构域等进行分析。结果表明:其开放阅读框长度为753 bp,编码250个氨基酸,蛋白分子量预测为27.41087 kDa,理论等电点为5.52,无跨膜螺旋区,属于稳定亲水性蛋白;氨基酸序列比对分析表明,菠萝AcAPX1与番木瓜、香蕉、椰子、油棕的序列相似性达85%以上,对AcAPX1基因进行功能结构域分析表明其具有血红素结合位点、K^(+)结合位点及底物结合位点(超家族:cl00196),属于植物过氧化物酶超家族的成员。AcAPX1基因的克隆对于研究其在菠萝中抗氧化能力中的分子机制及黑心病的防治具有重要意义。研究结果为进一步探究菠萝AcAPX1基因参与AsA清除ROS,延缓黑心病恶化的分子机制奠定�Internal browning(IB)seriously restricts the development of pineapple industry.Its occurrence is mainly from excessive reactive oxygen species(ROS)causing membrane damage,which breaks the original compartmentaliza-tion between plastid polyphenol oxidase(PPO)and phenolic substrates in vacuoles.It polymerizes into quinones after contact with oxidation.The antioxidant clearance system is able to remove excess ROS and maintain the dynamic bal-ance of ROS in the body.In early time,our laboratory used ascorbic acid(AsA)which can effectively delay the dete-rioration of IB of‘Pairs’pieapple.But the reaction mechanism of AsA is not clear.Ascorbate peroxidases(APX,EC1.1.11.1)are heme-binding enzymes that reduce hydrogen peroxide(H2O2)to water using AsA as an electron donor.It is involved in various developmental physiological processes and stress responses in plants.In this study,we selected six APX genes with AsA as the substrate in pineapple to analyze the occurrence process and the ascorbate transcript level changes after the AsA treatment.AcAPX1 was significantly upregulated at 9 d and 12 d after AsA treatment and was significantly different from the control group.It showed that AsA significantly enhanced the AcAPX1 expression.Correlation analysis showed that the incidence index of black heart disease was significantly positively correlated with the AcAPX1 expression level.It is speculated that this gene may play an important role in AsA delaying IB with pine-apple.To further explore the mechanism of action,AcAPX1 was cloned from the pineapple pulp.The physicochemical properties and protein domain were analyzed by bioinformatics analysis.The results showed that the open reading frame length was 753 bp,encoding 250 amino acids.The predicted molecular weight of the protein was 27.41087 kDa,and the theoretical isoelectric point was 5.52.There was no transmembrane helix region,which was a stable hydrophilic protein.Amino acid sequence alignment analysis showed that AcAPX1 of pineapple shared more than 85%homology with

关 键 词：菠萝 黑心病 抗坏血酸过氧化物酶 qPCR 克隆 

分 类 号：S436.68[农业科学—农业昆虫与害虫防治]