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植物经常暴露于各种环境(如干旱、盐碱、重金属等)胁迫中,严重影响了它们的酶和非酶成分,还对它们的相关基因构成威胁,且减缓了同化系统建成速度,影响生长进程。活性氧(ROS)在调节各种生物现象中起着重要的作用,包括激活细胞因子信号转导通路以及由此引起的基因表达。持续暴露于ROS中会引起植物的氧化胁迫,影响细胞内氧化还原平衡。非生物胁迫可导致细胞ROS浓度增加,随后转化为H2O2,它能够跨膜自由扩散,当其在细胞中的浓度积累到一定程度时,就会造成细胞损伤。同样,H2O2也可作为信号分子参与到胁迫信号转导途径中[1]。此外,活性氧不足可以引起细胞氧化胁迫,影响基因组的稳定性[2],因此氧化胁迫是细胞损伤的重要原因。蒋景龙等[3]施加不同浓度H2O2处理7 d苗龄的山黧豆幼苗24 h,分析山黧豆根系受氧化胁迫的程度与抗氧化系统的响应机制,结果表明,H2O2的积累与其受氧化胁迫程度呈正相关,且低浓度处理可以提高山黧豆抗氧化性能。Wan等[4]通过对12 d苗龄的水稻进行不同梯度H2O2处理6 h,分析叶片生理生化响应,并结合蛋白质组学揭示了水稻叶片生理特征变化与其胁迫响应蛋白之间的关系。
脱落酸(ABA)在植物非生物胁迫中起关键作用,它是激发植物应对不利环境条件的重要信号[5-6],并且能够协同调节胁迫反应中多种生理功能,包括气孔闭合,积累渗透调节物质和诱导胁迫相关蛋白的合成,如热休克蛋白,ROS清除剂等。然而,虽然许多非生物胁迫诱导基因受ABA控制,但有一些不是,这表明ABA依赖和非依赖性途径共同参与胁迫信号的传导[7-8]。ABA也是长距离信号分子,当环境条件发生变化时,它能够从成熟叶片持续地传输到发育叶片[9]。胁迫引起H2O2含量增加,造成氧化胁迫,而施加外源ABA,通过其信号转导,诱导相关基因表达,从而提高植物抗性[10-11]。Desikan等[12]研究发现,施加外源ABA有利于氧化胁迫下植物的适应生长,增加抗氧化酶的活性并诱导ROS清除系统等相关基因表达。王允等[13]研究表明,ABA可通过提高姜叶片ROS抗氧化系统的酶和非酶成分以抵御干旱引起的氧化胁迫。另外,研究显示外源物的调控效果与植物种类以及施用的方法、时间和浓度关系紧密[14, 15]。侧柏(Platycladus orientalis (Linn.) Franca)资源丰富,是生态环境修复的主要造林树种,具有一定的耐寒、耐旱、抗盐碱等特性,广泛分布于我国各地区,是我国特色树种,目前关于外源ABA与侧柏氧化胁迫交互作用的研究尚无报道。本试验研究了氧化胁迫下侧柏活性氧代谢以及施加外源ABA对其产生的作用,以期从生理生化和分子方面探讨侧柏的抗氧化胁迫和ABA的调控机制,为其更好地推广利用提供理论依据。
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试验是在预备试验的基础上,于2016年在中国林业科学研究院温室大棚内进行。以侧柏幼苗为试材,其种子采集于中国林业科学研究院院内,经浸种处理后,于2016年1月播种于塑料穴盆中,待生长至4月下旬,选择生长一致的植株,将其冲洗干净,转移至含有1/4 Hoagland溶液(pH 6.0)容器(10 L,60株/容器)中培育两周,待幼苗恢复正常生长,对侧柏进行试验处理。根据前期预实验结果,选择100 mmol·L-1 H2O2为胁迫处理浓度。试验处理具体如下:CK(1/4 Hoagland溶液)、CK+ABA0.5(0.5 μmol·L-1 ABA+1/4 Hoagland溶液)、CK+ABA200(200 μmol·L-1 ABA+1/4 Hoagland溶液)、H2O2(100 mmol·L-1 H2O2+1/4 Hoagland溶液)、H2O2+ABA0.5(0.5 μmol·L-1 ABA+100 mmol·L-1 H2O2+1/4 Hoagland溶液)、H2O2+ABA 200(200 μmol·L-1 ABA+100 mmol·L-1 H2O2+1/4 Hoagland溶液)。以上实验每处理3个生物学重复,每个生物学重复20株。所有植株处理6和48 h后取样,且进行液氮快速冷冻并存储在-80℃,为后续RNA提取和生理指标测定分析做准备。
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H2O2含量测定采用四氯化钛沉淀法[16];MDA含量测定采用硫代巴比妥酸显色法[17];SOD活性测定采用氮蓝四唑(NBT)法[18];POD活性测定方法参照Cakmak和Marschner [19];CAT活性测定方法参照Jablonski和Anderson [20]方法;GSH含量测定方法参照Anderson [21];Proline含量测定采用茚三酮比色法[22];可溶性蛋白测定采用考马斯亮蓝法[23]。
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采用植物RNA提取试剂盒(Tiandz)提取侧柏叶片的总RNA,并使用PrimeScriptTM RT reagent Kit(Takara)试剂盒反转录合成cDNA。根据侧柏转录组的注释Unigene,采用Primer Premier 3.0软件进行侧柏活性氧代谢相关基因定量引物设计,引物扩增效率均在95%~105%,PCR扩增产物均在100~150 bp,引物序列见表 1,αTUB为侧柏内参基因[24],相对表达量使用2-ΔΔCT方法[25],3个生物学重复。实时荧光定量PCR(qRT-PCR)测定在Roche LightCycler®480进行,使用SYBR®Premix Ex TaqTM II (Takara)试剂盒。PCR反应体系(20 μL)含有10 μL SYBR® primer Ex Taq(2×),每条引物0.8 μL(10 μmol·L-1),cDNA稀释13倍,取2.0 μL和6.4 μL蒸馏水。qRT-PCR反应程序为:95℃预变性10 s;95℃变性15 s,60℃退火30 s,40个循环。反应结束后对扩增产物荧光值变化和熔解曲线进行分析。
表 1 活性氧代谢相关基因引物
Table 1. The primers of genes related to reactive oxygen metabolism used for qRT-PCR
基因名称Gene name 引物Primer sequence (5'→3') Cu-Zn超氧化物歧化酶
(Cu/Zn-SOD) Superoxide
dismutase, Cu-Zn familyTTGAGGGCGTTGTGAGTCTC (Forward)
ACCTGTTGACATGCACCCAT (Reverse)过氧化氢酶(CAT)
CatalaseTTGTGAAACGTTGGGTGGGA (Forward)
CTTCTGGCCGAGGGATTTGT (Reverse)抗坏血酸盐过氧化物酶
(APX)
L-ascorbate peroxidaseGGGCTAACAGTGGCTTGGAT (Forward)
ACCTCAACAGCCACAACTCC (Reverse)谷胱甘肽还原酶(GR)
Glutathione reductaseTTGGAGCAGTGGGAGTTGAC (Forward)
GTTACATCACCGACTGCCCA (Reverse)单脱氢抗坏血酸还原酶
(MDAR)Monodehydroascorbate
reductaseTGGGGAGCTTGCCATCATTT (Forward)
TGGAAACCTGGTAGTCGTGC (Reverse)谷胱甘肽S-转移酶(GST)
Glutathionine S-transferaseTTTTGGCCTTGCAACCCTTT (Forward)
GACAAGTCCCCTTTTCCCCA (Reverse) -
利用Microsoft Excel 2013软件进行数据处理及作图,所有统计分析采用SPSS19.0软件,采用One-Way ANOVA进行比较。各处理之间的显著性差异检验水平为P < 0.05。
外源ABA对短期H2O2胁迫下侧柏幼苗活性氧代谢及相关基因的影响
Reactive Oxygen Metabolism and Its Related Gene Expression in Platycladus orientalis under H2O2 Stress and Regulated by ABA
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摘要:
目的 研究外源ABA处理对H2O2胁迫下侧柏幼苗活性氧代谢系统的影响,探讨ABA调控侧柏氧化胁迫的可能作用机制。 方法 以侧柏幼苗为试验材料,采用水培方式,研究外施低浓度(0.5 μmol·L-1)和高浓度(200 μmol·L-1)ABA对100 mmol·L-1 H2O2胁迫下侧柏幼苗活性氧代谢的影响。 结果 (1) 100 mmol·L-1 H2O2胁迫48 h显著增加了侧柏幼苗叶片过氧化氢(H2O2)、丙二醛(MDA)、谷胱甘肽(GSH)和脯氨酸含量、抗氧化物酶(SOD和CAT)活性,而可溶性蛋白含量降低。(2)相较于高浓度200 μmol·L-1 ABA,施加0.5 μmol·L-1 ABA显著减少了H2O2胁迫下侧柏幼苗H2O2和MDA的积累,进一步提高了侧柏幼苗叶片SOD、POD和CAT活性,同时促进GSH、脯氨酸和可溶性蛋白的合成。(3)100 mmol·L-1 H2O2胁迫处理48 h,侧柏幼苗叶片活性氧代谢相关基因Cu/Zn-SOD、CAT、GR、APX、MDAR和GST表达水平较对照CK均有显著性提高;正常和H2O2胁迫下侧柏幼苗外施0.5 μmol·L-1 ABA相较于200 μmol·L-1更有利于提高侧柏叶片活性氧代谢相关基因Cu/Zn-SOD、CAT、GR、APX、MDAR和GST的表达量。 结论 低浓度0.5 μmol·L-1 ABA有效地增强抗氧化系统的防御能力,减弱幼苗的氧化胁迫和膜脂过氧化水平,从而降低活性氧对侧柏的伤害。 Abstract:Objective To investigate the mechanism of exogenous abscisic acid(ABA)on regulating oxidative stress of Platycladus orientalis, the effects of reactive oxygen metabolism were studied in the leaves of P. orientalis exposed to hydrogen peroxide (H2O2) stress with the application of different concentrations of ABA. Method P. orientalis seedlings were exposed to 100 μmol·L-1 H2O2 were treated with 0.5 and 200 μmol·L-1 ABA, and physiological indexes and expression levels of genes related to reactive oxygen metabolism were studied. Result (1) 100 mmol·L-1 H2O2 significantly increased the contents of H2O2, malondialdehyde (MDA), glutathione (GSH) and proline and activities of antioxidant enzymes SOD and CAT in P. orientalis leaves, while soluble protein content decreased at 48 h. (2) Compared with 200 μmol·L-1 ABA, 0.5 μmol·L-1 ABA significantly enhanced the activities of SOD, POD and CAT, and increased the contents of GSH, proline and soluble protein in H2O2-treated seedlings, accompanied by the reduction of H2O2 and MDA contents. (3) 100 mmol·L-1 H2O2 up-regulated the expression levels of Cu/Zn-SOD、CAT、APX、MDAR和GST genes in P. orientalis at 48 h, and moreover, the presence of 0.5 μmol·L-1 ABA further prompted the expression levels of Cu/Zn-SOD、CAT、GR、APX、MDAR and GST genes, compared with 200 μmol·L-1 ABA under normal and H2O2 conditions. Conclusion Low concentration of 0.5 μmol·L-1 ABA effectively enhanced antioxidant defense, reduced oxidative stress and membrane lipid peroxidation in leaves of P. orientalis under H2O2 stress, which lowered the damage of reactive oxygen species to P. orientalis leaves and improved its resistance. -
Key words:
- Platycladus orientalis
- / H2O2 stress
- / ABA
- / reactive oxygen metabolism
- / gene expression
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表 1 活性氧代谢相关基因引物
Table 1. The primers of genes related to reactive oxygen metabolism used for qRT-PCR
基因名称Gene name 引物Primer sequence (5'→3') Cu-Zn超氧化物歧化酶
(Cu/Zn-SOD) Superoxide
dismutase, Cu-Zn familyTTGAGGGCGTTGTGAGTCTC (Forward)
ACCTGTTGACATGCACCCAT (Reverse)过氧化氢酶(CAT)
CatalaseTTGTGAAACGTTGGGTGGGA (Forward)
CTTCTGGCCGAGGGATTTGT (Reverse)抗坏血酸盐过氧化物酶
(APX)
L-ascorbate peroxidaseGGGCTAACAGTGGCTTGGAT (Forward)
ACCTCAACAGCCACAACTCC (Reverse)谷胱甘肽还原酶(GR)
Glutathione reductaseTTGGAGCAGTGGGAGTTGAC (Forward)
GTTACATCACCGACTGCCCA (Reverse)单脱氢抗坏血酸还原酶
(MDAR)Monodehydroascorbate
reductaseTGGGGAGCTTGCCATCATTT (Forward)
TGGAAACCTGGTAGTCGTGC (Reverse)谷胱甘肽S-转移酶(GST)
Glutathionine S-transferaseTTTTGGCCTTGCAACCCTTT (Forward)
GACAAGTCCCCTTTTCCCCA (Reverse) -
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