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

Abstract

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

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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Cloning, Sequence and Expression Analysis of Phosphoenolpyruvate Carboxykinase Genes (PEPCKs) in Apple

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