高山冰缘植物高山离子芥细胞磷脂酶D基因克隆及功能分析
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摘要
本研究以高山冰缘植物中的高山离子芥(Chorispora bungeana Fisch et. Mey, Chorispora bungean)作为研究材料,克隆了高山离子芥细胞磷脂酶D基因,通过RT-PCR、Western-blotting等方法研究了磷脂酶D在高山离子芥抵御胁迫过程中的作用,探讨了高山冰缘植物胁迫适应性与磷脂酶D的关系,揭示磷脂酶D在植物抗逆反应中的生理生化和分子生物学机制。取得的主要研究结果如下：
(1)克隆获得了高山离子芥细胞磷脂酶D基因(CbPLD),并分析预测了其结构组成,揭示了CbPLD的系统进化关系。结果显示：CbPLD cDNA全长2939bp,5’和3’端的非翻译区分别为70bp和39bp；含有一个完整的开放阅读框2712bp,编码903个氨基酸,蛋白质分子量约为100.5kDa,等电点为7.12。二级结构分析预测认为：CbPLD含有C2结构域、"IYIENQYF"结构域、以及磷脂酶D家族的活性结构域——两个HKD结构域；CbPLD预测蛋白序列包含26.14%α-螺旋(alpha helix),19.38%伸展链(extended strand),5.54%β-转角(beta turn),48.95%无规卷曲(random coil),其中α-螺旋和无规卷曲是CbPLD蛋白质中的主要二级结构域。从系统进化分析可以看出,CbPLD属于植物PLD beta亚型,在PLD beta亚型中,Chorispora bungeana和Arabidopsis PLD亲缘关系最接近,达到85%。
(2)研究了高山离子芥磷脂酶D基因CbPLD表达的组织特异性以及不同胁迫处理(温度、ABA、盐、以及H2O2)条件下转录和蛋白水平的变化,从转录和蛋白水平揭示了CbPLD可以被多种环境胁迫诱导,它广泛参与了高山离子芥对逆境胁迫的响应。结果表明：CbPLD表达不具备组织特异性,它是植物的生长和发育的基础因子之一；低温胁迫条件下,在-4℃下处理时,CbPLD基因的转录水平逐渐增长,并且在处理6天时达到峰值,之后逐渐下降；在4℃处理下,CbPLD基因的转录水平迅速增长,并在处理1天达到峰值,并在之后保持较高的水平,处理12天后表达量降低；Western-blot反应,杂交得到了分子量约为100kDa的主要特异性条带,该条带的明亮度与胁迫处理之间存在相关性,CbPLD蛋白质水平变化趋势与CbPLD基因的转录水平基本一致,推测认为,转录水平的增加导致了蛋白水平的增加；其他胁迫(ABA、盐、H2O2以及热胁迫)处理均诱导了CbPLD的表达。
(3)进行了高山离子芥细胞磷脂酶D响应冷冻胁迫的生化特性研究,阐明了高山离子芥细胞磷脂酶D响应冷冻胁迫的生化特性变化规律,揭示了磷脂酶D在高山离子芥特殊抗寒机制中的作用。结果显示：-4℃冷冻胁迫3天后,线粒体和微粒体膜结合态磷脂酶D酶活性显著增加,在6天时达到最高的酶活水平,之后逐渐下降。RT-PCR分析说明磷脂酶D基因转录水平变化与酶活性变化基本一致。冷冻胁迫下,线粒体和微粒体膜结合Ca2+含量均下降。酶动力学分析认为,线粒体和微粒体膜结合态磷脂酶D的活性遵从Michaelis-Menten酶动力学方程,冷冻胁迫分别引起线粒体和微粒体膜结合态磷脂酶D的Km和Vmax增加；酶活反应的最适pH和Ca2+含量发生了改变。
(4)研究了冷冻胁迫条件下,高山离子芥细胞磷脂酶D参与生理生化代谢的调控作用,分析了高山离子芥细胞磷脂酶D与膜稳定性、渗透调节物质积累和抗氧化酶系的关系,揭示了高山离子芥细胞磷脂酶D响应冷冻胁迫的生理生化代谢机制。结果表明,磷脂酶D参与了膜稳定性调节、渗透调节物质的积累和抗氧化酶系中过氧化氢酶活性的调控,并且与激素ABA信号通路相关。
Chorispora bungeana Fisch. and C.A. Mey (Chorispora bungeana) is a rare alpine subnival plant species that is highly tolerant of stress environment. Phospholipase D (PLD) is a key enzyme involved in membrane phospholipid catabolism during plant growth, development and stress responses. We have isolated and partially characterized a full-length cDNA encoding PLD from the calluses of Chorispora bungeana, with the aim of furthering our understanding of the role of PLD at the molecular level. Although PLD has been studied in many plants, no examinations of its metabolism, regulation and molecular properties during environmental stresses have been undertaken. In this study, we investigated the response of PLD to environmental stresses and the role of PLD in metabolism pathway of Chorispora bungeana. The main results are as follows:
(1) Through PCR and RACE techniques, a whole sequence of PLD cDNA was isolated from Chorispora bungeana (GenBank accession No. HM756247). The cloned full-length cDNA of Chorispora bungeana (CbPLD) was2939bp. The cDNA contained a2712bp ORF encoding a protein of903amino acids with a calculated molecular weight of about100.5kDa and with a PI of7.12. A5'untranslated region (UTR) of70bp was found upstream of the first ATG codon, and a3'untranslated region (UTR) of39bp was found downstream from the stop codon. Secondary structure analysis showed that CbPLD had C2domain,"IYIENQYF" region and two HKD motifs belonging to PLD family as their activity region. The putative CbPLD peptide contained26.14%alpha helix,19.38%extended strand,5.54%beta turn, and48.95%random coil. The alpha helix and random coil constituted interlaced domination of the main part of the secondary structure.A comparison of the predicted protein sequences of the CbPLD with PLDs of other plants shows that CbPLD is highly identical to PLD from Arabidopsis thaliana PLD beta (85%).
(2) CbPLD expression patterns under stresses were analyzed by semi-quantitative RT-PCR. The results indicated that CbPLD is ubiquitously expressed in Chorispora bungeana, with almost no tissue specificity even though the expression levels showed slight variations between tissue types. The accumulation pattern of the CbPLD mRNA in response to low temperature was studied. Following exposure to-4℃, the transcript levels of CbPLD progressively increased, almost reaching the peak at6days of treatment, and then declined. After exposure to4℃, the transcript level of CbPLD increased rapidly. The level of the CbPLD transcript almost reached the peak at1day and remained at a high level, then decreased at12days. Immunoblots of PLD protein showed a molecular mass around100kDa. In parallel with the RT-PCR and immunoblots analyses, there were no distinguishable differences. The increases of PLD protein content are corresponding to the different levels of CbPLD mRNA. It seems to be a clear correlation between increase in CbPLD transcription levels and protein contents at different days of low temperature treatments. These results suggested that the major cause of the increases of PLD protein content during the low temperature stresses in Chorispora bungeana callus could be due to the up-regulation of CbPLD gene.
The transcripts of CbPLD accumulated highly when Chorispora bungeana was treated with abscisic acid (ABA), salinity, hydrogen peroxide (H2O2) and heat. These results indicate that the CbPLD may play an important role in response to stresses in Chorispora bungeana.
(3) The influence of freezing treatment on CbPLD activities was studied in Chorispora bungeana. The results showed that:
During the freezing treatment (-4℃), PLD activities in both microsomal and mitochondrial membranes showed significant increases at day3, and remained at a high level at day6, then declined to a moderate level. The RT-PCR analyses showed that PLD activity partially corresponded to the PLD gene transcript level. Freezing injury, as measured by electrolyte leakage and malondialdehyde content, peaked at day6and then gradually decreased to a low level. Alleviation of freezing injury was related to a decreased content of membrane-associated Ca2+and in parallel with the changes of PLD activities. The influence of freezing treatment on PLD catalytic mechanism showed that freezing treatment resulted in increases in the Km and Vmax for microsomal and mitochondrial PLD, respectively. Both the optimal pH and calcium ion concentration of the mitochondrial and microsomal PLD were changed under freezing treatment. The findings indicated that PLD is involved in membrane deterioration and the signaling pathway in response to freezing stress, and the specific mechanism of cold resistance of Chorispora bungeana is linked with PLD.
(4) The metabolism regulations of CbPLD in response to freezing stress were analyzed. The results showed that CbPLD have mediated the regulation pathway of membrane stability, osmotic adjustment and regulated CAT activity in anti-oxidative system. It also involved in the pathway of ABA signaling.
(1)克隆获得了高山离子芥细胞磷脂酶D基因(CbPLD),并分析预测了其结构组成,揭示了CbPLD的系统进化关系。结果显示：CbPLD cDNA全长2939bp,5’和3’端的非翻译区分别为70bp和39bp；含有一个完整的开放阅读框2712bp,编码903个氨基酸,蛋白质分子量约为100.5kDa,等电点为7.12。二级结构分析预测认为：CbPLD含有C2结构域、"IYIENQYF"结构域、以及磷脂酶D家族的活性结构域——两个HKD结构域；CbPLD预测蛋白序列包含26.14%α-螺旋(alpha helix),19.38%伸展链(extended strand),5.54%β-转角(beta turn),48.95%无规卷曲(random coil),其中α-螺旋和无规卷曲是CbPLD蛋白质中的主要二级结构域。从系统进化分析可以看出,CbPLD属于植物PLD beta亚型,在PLD beta亚型中,Chorispora bungeana和Arabidopsis PLD亲缘关系最接近,达到85%。
(2)研究了高山离子芥磷脂酶D基因CbPLD表达的组织特异性以及不同胁迫处理(温度、ABA、盐、以及H2O2)条件下转录和蛋白水平的变化,从转录和蛋白水平揭示了CbPLD可以被多种环境胁迫诱导,它广泛参与了高山离子芥对逆境胁迫的响应。结果表明：CbPLD表达不具备组织特异性,它是植物的生长和发育的基础因子之一；低温胁迫条件下,在-4℃下处理时,CbPLD基因的转录水平逐渐增长,并且在处理6天时达到峰值,之后逐渐下降；在4℃处理下,CbPLD基因的转录水平迅速增长,并在处理1天达到峰值,并在之后保持较高的水平,处理12天后表达量降低；Western-blot反应,杂交得到了分子量约为100kDa的主要特异性条带,该条带的明亮度与胁迫处理之间存在相关性,CbPLD蛋白质水平变化趋势与CbPLD基因的转录水平基本一致,推测认为,转录水平的增加导致了蛋白水平的增加；其他胁迫(ABA、盐、H2O2以及热胁迫)处理均诱导了CbPLD的表达。
(3)进行了高山离子芥细胞磷脂酶D响应冷冻胁迫的生化特性研究,阐明了高山离子芥细胞磷脂酶D响应冷冻胁迫的生化特性变化规律,揭示了磷脂酶D在高山离子芥特殊抗寒机制中的作用。结果显示：-4℃冷冻胁迫3天后,线粒体和微粒体膜结合态磷脂酶D酶活性显著增加,在6天时达到最高的酶活水平,之后逐渐下降。RT-PCR分析说明磷脂酶D基因转录水平变化与酶活性变化基本一致。冷冻胁迫下,线粒体和微粒体膜结合Ca2+含量均下降。酶动力学分析认为,线粒体和微粒体膜结合态磷脂酶D的活性遵从Michaelis-Menten酶动力学方程,冷冻胁迫分别引起线粒体和微粒体膜结合态磷脂酶D的Km和Vmax增加；酶活反应的最适pH和Ca2+含量发生了改变。
(4)研究了冷冻胁迫条件下,高山离子芥细胞磷脂酶D参与生理生化代谢的调控作用,分析了高山离子芥细胞磷脂酶D与膜稳定性、渗透调节物质积累和抗氧化酶系的关系,揭示了高山离子芥细胞磷脂酶D响应冷冻胁迫的生理生化代谢机制。结果表明,磷脂酶D参与了膜稳定性调节、渗透调节物质的积累和抗氧化酶系中过氧化氢酶活性的调控,并且与激素ABA信号通路相关。
Chorispora bungeana Fisch. and C.A. Mey (Chorispora bungeana) is a rare alpine subnival plant species that is highly tolerant of stress environment. Phospholipase D (PLD) is a key enzyme involved in membrane phospholipid catabolism during plant growth, development and stress responses. We have isolated and partially characterized a full-length cDNA encoding PLD from the calluses of Chorispora bungeana, with the aim of furthering our understanding of the role of PLD at the molecular level. Although PLD has been studied in many plants, no examinations of its metabolism, regulation and molecular properties during environmental stresses have been undertaken. In this study, we investigated the response of PLD to environmental stresses and the role of PLD in metabolism pathway of Chorispora bungeana. The main results are as follows:
(1) Through PCR and RACE techniques, a whole sequence of PLD cDNA was isolated from Chorispora bungeana (GenBank accession No. HM756247). The cloned full-length cDNA of Chorispora bungeana (CbPLD) was2939bp. The cDNA contained a2712bp ORF encoding a protein of903amino acids with a calculated molecular weight of about100.5kDa and with a PI of7.12. A5'untranslated region (UTR) of70bp was found upstream of the first ATG codon, and a3'untranslated region (UTR) of39bp was found downstream from the stop codon. Secondary structure analysis showed that CbPLD had C2domain,"IYIENQYF" region and two HKD motifs belonging to PLD family as their activity region. The putative CbPLD peptide contained26.14%alpha helix,19.38%extended strand,5.54%beta turn, and48.95%random coil. The alpha helix and random coil constituted interlaced domination of the main part of the secondary structure.A comparison of the predicted protein sequences of the CbPLD with PLDs of other plants shows that CbPLD is highly identical to PLD from Arabidopsis thaliana PLD beta (85%).
(2) CbPLD expression patterns under stresses were analyzed by semi-quantitative RT-PCR. The results indicated that CbPLD is ubiquitously expressed in Chorispora bungeana, with almost no tissue specificity even though the expression levels showed slight variations between tissue types. The accumulation pattern of the CbPLD mRNA in response to low temperature was studied. Following exposure to-4℃, the transcript levels of CbPLD progressively increased, almost reaching the peak at6days of treatment, and then declined. After exposure to4℃, the transcript level of CbPLD increased rapidly. The level of the CbPLD transcript almost reached the peak at1day and remained at a high level, then decreased at12days. Immunoblots of PLD protein showed a molecular mass around100kDa. In parallel with the RT-PCR and immunoblots analyses, there were no distinguishable differences. The increases of PLD protein content are corresponding to the different levels of CbPLD mRNA. It seems to be a clear correlation between increase in CbPLD transcription levels and protein contents at different days of low temperature treatments. These results suggested that the major cause of the increases of PLD protein content during the low temperature stresses in Chorispora bungeana callus could be due to the up-regulation of CbPLD gene.
The transcripts of CbPLD accumulated highly when Chorispora bungeana was treated with abscisic acid (ABA), salinity, hydrogen peroxide (H2O2) and heat. These results indicate that the CbPLD may play an important role in response to stresses in Chorispora bungeana.
(3) The influence of freezing treatment on CbPLD activities was studied in Chorispora bungeana. The results showed that:
During the freezing treatment (-4℃), PLD activities in both microsomal and mitochondrial membranes showed significant increases at day3, and remained at a high level at day6, then declined to a moderate level. The RT-PCR analyses showed that PLD activity partially corresponded to the PLD gene transcript level. Freezing injury, as measured by electrolyte leakage and malondialdehyde content, peaked at day6and then gradually decreased to a low level. Alleviation of freezing injury was related to a decreased content of membrane-associated Ca2+and in parallel with the changes of PLD activities. The influence of freezing treatment on PLD catalytic mechanism showed that freezing treatment resulted in increases in the Km and Vmax for microsomal and mitochondrial PLD, respectively. Both the optimal pH and calcium ion concentration of the mitochondrial and microsomal PLD were changed under freezing treatment. The findings indicated that PLD is involved in membrane deterioration and the signaling pathway in response to freezing stress, and the specific mechanism of cold resistance of Chorispora bungeana is linked with PLD.
(4) The metabolism regulations of CbPLD in response to freezing stress were analyzed. The results showed that CbPLD have mediated the regulation pathway of membrane stability, osmotic adjustment and regulated CAT activity in anti-oxidative system. It also involved in the pathway of ABA signaling.
引文
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Shaterian J, Georges F, Hussain A, Waterer D, De Jong H, Tanino KK. Root to shoot communication and abscisic acid in calreticulin (CR) gene expression and salt-stress tolerance in grafted diploid potato clones. Environ Exp Bot,2005,53:323-332
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Srivastava S, Rahman MH, Shah S, Kav NNV. Constitutive expression of the pea ABA-responsive 17 (ABR17) cDNA confers multiple stress tolerance in Arabidopsis thaliana. Plant Biotech J,2006,4:529-549
Sun WH, Duan M, Li F, Shu DF, Yang S, Meng QW. Over expression of tomato t APX gene in tobacco improves tolerance to high or low temperature stress. Biol Plant, 2010,54(4):614-620
Sung TC, Roper RL, Zhang Y. Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein requried for poxvirus pathogenicity. ENBO, 1997,16:4519-4530
Thompson J E. The molecular basis for membrane deterioration during senescence, In: Jandus J. Phospholipase D during tomato fruit ripening. Plant Physiol Biochem, 1997,35:123-128
Uemura M, Steponkus PL. Cold acclimation in plants:relationship between the lipid composition and the cryostability of the plasma membrane. J. Plant Res,1999,112, (2):245-254
Uike J, Morioka S, Komari T. Purification and characterization of phospholipase D(PLD) from fice and cloning of cDNA for phospholipase D from rice and maize.Plant Cell Physiol,1995,36:903-914
Ulbrich-Hofmann R, Lerchner A, Oblozinsky & Lydia Bezakova M. Phospholipase D and its application in biocatalysis. Biotechnology Letters,2005,27:535-543
Walker DJ, Romero P, Correal E. Cold tolerance, water relations and accumulation of osmolytes in Bituminaria bituminosa. Biol Plant,2010,54(2):293-298
Wan SB, Tian L, Tian R R, Pan QH, Zhan JC, Wen PF, Chen JY, Zhang P, Wang W, Huang WD. Involvement of phospholipase D in the low temperature acclimation-induced thermotolerance in grape berry. Plant Physiology and Biochemistry,2009,47:504-510
Wang LJ, Huang WD, Li JY, Liu YF, Shi YL. Peroxidation of membrane lipid and Ca2 homeostasis in grape mesophyll cells during the process of cross adaptation to temperature stresses. Plant Sci,2004,167:71-77
Wang XM, Xu L, Zheng L. Cloning and expression of phosphatidylcholine-hydrolyzing phospholipase D from Ricinus communis L..J Bio Chem,1994,269(32):20312-20317
Wang XM. The role of phospholipase D in signaling cascades.Plant Physiology,1999, 120:645-656
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Wang XM.Plant phospholipases.Annu.Rev.Plant Physiol.Plant Mol.Biol,2001,52:211-231
Wang XM. Phospholipase D in hormonal and stress signaling. Current Opinion in Plant Biology,2002,5:408-414
Wang XM,Wang CX, Sang YM,Qin CB,Welti R.Networking of phospholipases in plant signal transduction.Physiol.Planta.,2002,115:331-335
Wang XM. Lipid signaling. Current Opinion in Plant Biology,2004,7:329-336
Wu JM, Qu T, Chen SY, Zhao ZG, An LZ.Molecular cloning and characterization of a Y-glutamylcysteine synthetase gene from Chorispora bungeana.Protoplasma,2009,235: 27-36
Xiong L, Schumaker KS, Zhu JK. Cell signaling during cold, drought, and salt stress. Plant Cell,2002,14:165-183
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