苹果磷酸烯醇式丙酮酸羧化激酶基因（PEPCKs）的克隆与表达分析

doi:10.3864/j.issn.0578-1752.2016.07.017

摘要/Abstract

摘要： 【目的】克隆苹果（Malus×domestica Borkh.）多个代谢途径中的关键酶磷酸烯醇式丙酮酸羧化激酶（phosphoenolpyruvate carboxykinase，PEPCK）基因MdPCKs，研究其在不同组织器官及不同胁迫处理条件下的表达特性，为解析该基因在多个代谢途径中的功能奠定基础。【方法】利用同源比对和RT-PCR技术，克隆获得MdPCK1和MdPCK2全长cDNA序列并进行生物信息学分析；克隆MdPCKs的启动子序列，利用PlantCARE软件在线分析启动子上的顺式作用元件；采用实时荧光定量PCR（qRT-PCR）检测MdPCKs在不同组织中的表达以及在水杨酸（SA）、茉莉酸（JA）、脱落酸（ABA）、模拟干旱（PEG）、高温（40℃）和低温（8℃）处理条件下的表达特性。【结果】获得2个苹果磷酸烯醇式丙酮酸羧化激酶基因（MdPCK1和MdPCK2；GenBank登录号分别为KT454964和KT454965），其开放阅读框（open reading frame，ORF）分别为2 001 bp和2 028 bp，分别编码666和675个氨基酸残基；基因结构和进化分析表明，MdPCK1和MdPCK2均由12个外显子组成，且序列结构进化高度保守；氨基酸序列和结构分析显示，二者均含有保守的PEPCK ATP domain区域；启动子分析显示，MdPCK1和MdPCK2启动子区域含有多个顺式作用元件，包括光响应元件、昼夜节律相关顺式作用元件、JA响应元件、SA响应元件、ABA响应元件、防御及逆境响应元件、低温响应元件和热激响应元件等；qRT-PCR结果显示，MdPCKs在被检测的组织中均有表达。在‘泰山嘎啦’苹果果实不同发育阶段，MdPCK1和MdPCK2具有相似的表达模式。在多种非生物胁迫处理下，MdPCK1受到100 μmol·L^-1 ABA和100 g·L^-1 PEG胁迫诱导；MdPCK2受到150 μmol·L^-1 SA、100 μmol·L^-1 ABA、100 g·L^-1 PEG和低温胁迫诱导。【结论】MdPCK1和MdPCK2属于植物PEPCK家族，非生物逆境胁迫可诱导MdPCK1和MdPCK2表达。

关键词: 苹果, 磷酸烯醇式丙酮酸羧化激酶, 序列分析, 胁迫诱导, 表达分析

Abstract: 【Objective】The aim of this study is to characterize an ubiquitous enzyme of phosphoenolpyruvate carboxykinase (PEPCK) involved in many pathways of plant metabolism. The transcriptional level of MdPCKs in different tissues and under various stress treatments were determined to predict the related function of MdPCKs gene. 【Method】 The full-length cDNA sequences of MdPCK1 and MdPCK2 were isolatedby homologous alignment and RT-PCR confirmation, the obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods; the promoters of MdPCK1 and MdPCK2 were cloned and the cis-acting regulatory elements were analyzed through PlantCARE database; the expression levels of MdPCK1 and MdPCK2 were detected in different tissues and under SA, JA, ABA, PEG, high temperature and low temperature treatments using qRT-PCR.【Result】The sequencing results showed that two cDNAs (designated as MdPCK1 and MdPCK2; GenBank Accession No. KT454964 and KT454965) contained an open reading frame (ORF) of 2 001 bp and 2 028 bp, and their ORFs encoded a protein with 666 and 675 amino acids, respectively. The results of gene structure analysis revealed that both of the MdPCK1 and MdPCK2 contained 12 exons and PEPCK gene structures were highly conserved in plant. Amino acid sequences and structure analysis indicated that both of the MdPCK1 and MdPCK2 contained conserved PEPCK ATP domain. The results of promoter analysis showed that there were multiple putative cis-acting elements involved in light responsiveness, circadian control, MeJA-responsiveness, salicylic acid responsiveness, abscisic acid responsiveness, defense and stress responsiveness, low- temperature responsiveness and heat stress responsiveness. qRT-PCR results showed that MdPCKs were constitutively expressed in all examined tissues. MdPCK1 and MdPCK2 had similar expression patterns during fruit development of ‘Taishan Gala’ apple. Under various stress conditions, MdPCK1 expression was induced by 100 μmol·L^-1ABA and 100 g·L^-1 PEG, and MdPCK2 expression was induced by 150 μmol·L^-1 SA, 100 μmol·L^-1ABA, 100 g·L^-1 PEG and low temperature.【Conclusion】Taken together, the above results indicated that MdPCK1 and MdPCK2 belonged to plant PEPCK family, their expressions could be induced by abiotic stresses.

Key words: apple, phosphoenolpyruvate carboxykinase, sequence analysis, stress induced, expression analysis

李慧峰，冉昆，程来亮，王海波，何平，常源升，李林光. 苹果磷酸烯醇式丙酮酸羧化激酶基因（PEPCKs）的克隆与表达分析[J]. 中国农业科学, 2016, 49(7): 1417-1428.

LI Hui-feng, RAN Kun, CHENG Lai-liang, WANG Hai-bo, HE Ping, CHANG Yuan-sheng, LI Lin-guang. Cloning, Sequence and Expression Analysis of Phosphoenolpyruvate Carboxykinase Genes (PEPCKs) in Apple[J]. Scientia Agricultura Sinica, 2016, 49(7): 1417-1428.

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