黄皮果实ClPPO基因克隆及表达分析  

Cloning and Expression Analysis of ClPPO Gene in Clausena lansium

作　　者：沈丸钧 刘新婷 刘照宇 李雯[1] 曾教科 Shen Wanjun;Liu Xinting;Liu Zhaoyu;Li Wen;Zeng Jiaoke(Key Laboratory for Quality Regulation of Tropical Horticulture Plants of Hainan Province,College of Horticulture,Hainan University,Haikou,570228;Key Laboratory for Postharvest Technology and Nondestructive Testing of Fruits&Vegetables of Jiangxi Province,School of Agricultural Sciences,Jiangxi Agricultural University,Nanchang,330045)

机构地区：[1]海南大学园艺学院,海南省热带园艺作物品质调控重点实验室,海口570228 [2]江西农业大学农学院,江西省果蔬保鲜与无损检测重点实验室,南昌330045

出　　处：《分子植物育种》2022年第19期6341-6349,共9页Molecular Plant Breeding

基　　金：海南省自然科学基金高层次人才项目(320RC488);海南省教育厅项目(Hnky2019-18)共同资助。

摘　　要：多酚氧化酶(polyphenol oxidase,PPO)是果实采后酶促褐变的一个关键酶。为了获得黄皮(Clausena lansium(Lour.)Skeels)中多酚氧化酶基因序列信息及其在低温贮藏期间的表达情况,基于本实验室前期转录组测序结果,通过TA克隆及RT-PCR(Reverse transcription PCR)技术,鉴定了一个PPO基因,命名为ClPPO(GenBank登录号:MW532091)。ClPPO全长1782 bp,具有完整的开放阅读框,编码593个氨基酸,包含Tyrosinase、PPO1_DWL和PPO1_KFDV三个保守结构域。与其他植物的氨基酸序列总体相似性为75.85%,与柑橘CcPPO进化关系最近。ClPPO为亲水不稳定脂类非分泌蛋白,无跨膜蛋白区,含有63个磷酸化位点和3个二硫键。ClPPO蛋白酶的二级结构无规则卷曲比例最高,三级空间结构模型与苹果络氨酸酶(MdPPO1,6els.1.A)序列同源性达70%以上。亚细胞定位分析推断ClPPO蛋白定位于叶绿体中。RT-qPCR实验表明,低温贮藏过程中,ClPPO基因表达量在果肉中呈先上升后下降的趋势,HT(热处理,50℃/30 s)和LTC(程序降温,8℃/4 d)处理均能有效抑制ClPPO基因表达的上升,ClPPO基因表达与果肉PPO酶活性变化趋势高度一致。果皮PPO酶活性呈现与果肉类似的变化趋势,但果皮ClPPO基因表达与PPO酶活性间变化趋势并不一致,表明ClPPO基因主要在黄皮果肉中起到调控作用,从而延缓果肉褐变及衰老进程。Polyphenol oxidase is the key enzyme of enzymatic browning for postharvest fruits.In order to acquire the sequence information and gene expression pattern of PPO in cold stored wampee fruit(Clausena lansium(Lour.)Skeels),we identified a polyphenol oxidase gene through RNA-Sequencing,TA cloning and RT-PCR(Reverse transcription PCR)technology,namely ClPPO(GenBank number:MW532091).The results elucidated that ClPPO contained an open reading frame of 1782 bp,which was intended to encode 593 amino acid residues.The PPO protein contained three conserved domains,i.e.Tyrosinase,PPO1_DWL and PPO1_KFDV.In addition,its total similarity was 75.85%,comparing to other plant PPO,and wampee has the closest evolutionary relationship with CcPPO(Citrus clementina).ClPPO is a hydrophilic and non-secretory lipoprotein.It is unstable and does not contain the transmembrane protein region,but ClPPO contains 63 phosphorylation sites and 3 disulfide bonds.The protein structure analysis predicted that random coils accounted for the highest propotion in the secondary structure of ClPPO,and the tertiary structure model indicated more than 70% sequence consistency between ClPPO and apple tyrosinase(MDPPO1,6els.1.A).Subcellular localization analysis suggested that ClPPO was localized in chloroplasts.We also investigated the temperature responses of ClPPO expression to HT(heat treatment,50℃/30 s)and LTC(low temperature conditioning,8℃/4 d)treatments using RT-qPCR.The results showed that ClPPO gene and PPO enzyme activity were up-regulated in the first 2 or 4 days of storage,and then decreased rapidly during cold storage in wampee flesh.HT and LTC treatments could effectively inhibit the ClPPO up-expression and PPO enzyme activity.In wampee peel,the PPO enzyme activity showed a similar trend to that of the pulp,but the correlation between the changes of ClPPO expression and PPO enzyme activity in peel was not consistent,indicating that the ClPPO gene mainly played a regulatory role in the wampee pulp browning during late storage period.

关 键 词：黄皮(Clausena lansium L.) 多酚氧化酶基因 基因克隆 表达分析 

分 类 号：S666.6[农业科学—果树学]