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在环境胁迫下，植物会产生大量的活性氧（ROS），主要有H2O2、超氧阴离子O2−和一氧化氮等[1]。此外，ROS也是重要的信号分子，参与调节植物的许多生长发育过程，包括细胞程序性死亡、激素作用、病原体反应、压力感知和光合作用调节等[1-5]。然而ROS起着双重作用，活性氧浓度过高会导致细胞遭受不可逆转的破坏，最终致死[1, 5-6]。
在植物细胞中存在两种系统共同维持着ROS的稳定。当细胞内产生高浓度的ROS时，首先，SOD参与毒性较强的O2−转化为毒性较弱的H2O2[7]，再通过CAT和抗坏血酸-谷胱甘肽循环（AsA-GSH cycle）将H2O2清除，APX参与将H2O2催化形成H2O的过程[8]。APX的表达受到盐胁迫、干旱胁迫、氧化胁迫等[9]多种环境刺激的调节，在这些逆境下，APX的活性明显增强[10]。
APX是一个多基因家族，定位在叶绿体基质、叶绿体类囊体、细胞质、微体以及线粒体[11-14]。不同细胞器中的H2O2主要是由自身的APX清除的[15-16]。小麦（Triticum aestivum）两个APX基因的表达受干旱胁迫的影响[17]，5个水稻APX基因OsAPX 1、OsAPX 2、OsAPX 5、OsAPX 6、OsAPX 7的表达量增加[18]，OsAPX 8的表达量呈现显著下降的趋势。受到盐胁迫时，水稻（Oryza sativa）OsAPX 2、OsAPX 7和OsAPX 8这3个APX基因转录水平的表达出现明显的差异[19]。在高温和干旱的双重胁迫下，拟南芥APX1在蛋白水平和转录水平的表达量都表现出明显上升趋势，APX1的突变体、野生型拟南芥植株与受胁迫植株相比，上升趋势更为明显[20]。在植株中过表达APX基因通常可以增加植株在逆境中的抗性。转水稻APX2基因的拟南芥植株在盐胁迫下表现出更强的耐受性[21-22]。拟南芥APX在烟草中过表达可以促进烟草植株的抗性[23]。
本研究取野生型烟草植株和过表达PtomtAPX基因的烟草植株分别进行以下3种非生物胁迫处理：盐胁迫、干旱胁迫和氧化胁迫。测定并记录处理过程中的生理数据的变化，比较野生型植株与PtomtAPX-OE烟草植株的差异，更加深入地了解PtomtAPX在逆境中对植物生长的影响。
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PtomtAPX-OE烟草植株由实验室前期工作所得[24]，培养条件为25 ℃，光照时间16 h，光照强度为250 μmol/（m2∙s）。取长势相似且状态良好的野生型烟草与PtomtAPX-OE作为实验材料，分别进行盐胁迫、氧化胁迫和干旱胁迫处理，处理条件如下。
对照植株浇水50 mL，干旱胁迫植株不浇水，盐胁迫植株用200 mmol/L NaCl含量的水浇灌50 mL，氧化胁迫植株是喷洒50 mmol/L甲基紫精（MV）溶液于烟草叶片上。
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取野生型和转基因烟草叶片，鲜质量记为Fw，饱和鲜质量用Tw表示，于80 ℃烘箱中烘干，干质量记为Dw。相对含水量RWC = （Fw − Dw）/（Tw − Dw） × 100%。
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选取PtomtAPX-OE和野生型烟草叶片，分别加入80%丙酮5 mL，DMSO 5 mL，水浴后冷却，定容至25 mL，空白对照为80%丙酮，分别测定OD663 nm和OD645 nm数值。
叶绿素含量 = Chlt × 0.025 × 2.5/W
Chlt = 8.02 × OD663 nm + 20.21 × OD645 nm
W = Fw（1 − RWC）
式中：W为叶片干质量，Chlt为叶绿素总含量。
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分别称量PtomtAPX-OE和野生型烟草叶片0.5 g，用液氮研磨，分别吸取625 μL 0.1% TCA和0.5% TBA溶液625 μL加入研磨液中，沸水加热10 min，常温冷却，用转速为8 000 r/min离心10 min；同时用0.5% TBA为空白对照，分别在OD660 nm和OD532 nm下进行测定吸光值。
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Bradford法测定总蛋白含量。
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取3 mL活性测定反应液（0.1 mmol/L EDTA， 50 mmol/L磷酸缓冲液，pH 7.0，0.5 mmol/L ASA，0.1 mmol/L H2O2）于试管中，加酶蛋白提取液50 μL。空白对照为不加AsA的反应液，每隔1 min记录1次OD290 nm，连续记录5次。
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取4 mL活性测定反应液（2.25 mmol/L NBT，0.1 mmol/L EDTA， 13 mmol/L Met，60 μmol/L核黄素，50 mmol/L pH 7.8的磷酸缓冲液）于试管中，加酶蛋白提取液20 μL，以未加酶液的磷酸缓冲液A0为空白对照，测定OD560 nm的变化。
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按照AsA试剂盒的说明书操作。
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采用试剂盒（amplex red hydrogen peroxide/peroxidase assay）测定H2O2含量。
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采用EnzyChrom NADP/NADPH Assay试剂盒测定NADP/NADPH的比值。
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干旱胁迫下，野生型植株与PtomtAPX-OE植株的含水量分别下降62.4%和47.4%；比较叶绿素含量发现PtomtAPX-OE植株是野生型植株的1.6倍；可溶性总蛋白明显下降；细胞内MDA的增加量为野生型的55.1%（图1）。
图 1 PtomtAPX-OE烟草与野生型烟草在干旱环境下的应答反应
Figure 1. Responses of overexpressed APX transgenic tobaccos and wild type under drought environment
在干旱胁迫下，H2O2含量及APX活性都有显著变化。PtomtAPX-OE植株的H2O2上升量为野生型的50.4%，APX活性的平均增加值是野生型的1.77倍。APX活性的上升会导致AsA消耗量的增加，在受到干旱胁迫后野生型植株AsA的消耗量为52.2%，而转基因植株的AsA消耗量为63.5%（图2）。
图 2 干旱胁迫下野生型与PtomtAPX-OE烟草的差异
Figure 2. Differences between wild type and APX-overexpressed tobaccos under drought stress
AsA的再生由NADPH作为电子供体，在受到干旱胁迫后，野生型植株与PtomtAPX-OE植株NADP/NADPH值、SOD值均出现了显著的上升，NADP/NADPH值分别升高1.18和3.36倍，转基因植株SOD值的升高值为野生型的1.81倍（图3）。
图 3 干旱胁迫下PtomtAPX-OE烟草与野生型其他生理应答的差异
Figure 3. Differences in physiological responses of APX-overexpressed tobacco and wild type under drought stress
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在受到盐胁迫后，野生型和PtomtAPX-OE植株的可溶性总蛋白、相对含水量及叶绿素含量下降，MDA含量、APX活性、NADP/NADPH比值、SOD酶活性出现了显著的上升。转基因植株中，可溶性总蛋白含量比野生型高23.1%，APX活性升高81.7%，SOD酶活性升高57.7%，MDA含量升高2.5倍，相对含水量下降22.2%，叶绿素含量降低为野生型的47.2%。在野生型植株中，MDA含量升高3.7倍，APX活性升高19%，SOD酶活性升高44.8%，相对含水量下降33.2%（图4）。
图 4 盐胁迫对PtomtAPX-OE烟草与野生型烟草叶片的影响
Figure 4. Effects of salt stress on APX-overexpressed tobacco and wild type tobacco leaves
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本研究发现，在氧化胁迫下，野生型植株NADP/NADPH升高64%，APX活性升高38.5%。在PtomtAPX-OE植株中，叶绿素含量为野生型的1.78倍。APX活性升高91%，相对含水量降低值比野生型低14.8%，SOD酶活性升高值比野生型高52%，MDA升高值为野生型的55.3%，可溶性总蛋白的降低值为野生型的28%，H2O2升高值仅为野生型的48.2%（图5）。
图 5 氧化胁迫对PtomtAPX-OE烟草与野生型植株的影响
Figure 5. Effects of oxidative stress on APX-overexpressed tobacco and wild type plants
与干旱胁迫和盐胁迫类似，植株在受到氧化胁迫后，PtomtAPX-OE和野生型植株的可溶性总蛋白、相对含水量及叶绿素含量大幅度下降，分别为野生型的28%、14.8%及1.78倍，MDA含量、APX活性、NADP/NADPH比值、SOD酶活性出现了显著的上升。野生型植株中，APX活性升高38.5%，NADP/NADPH比值升高64%。PtomtAPX-OE植株中，MDA升高值为野生型的55.3%，APX活性升高91%，NADP/NADPH比值升高2.48倍。SOD酶活性升高值比野生型高52%。
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有研究表明，胁迫环境中产生的ROS会严重影响植株的正常生长，其中细胞膜受损较为严重[25-27]。由于ROS的大量产生，活性氧消除体系的代谢效率与植株在逆境中的抗性紧密相关[28-30]。在植物逆境应答中，AsA对于维持APX清除H2O2的能力至关重要[31-32]。
本研究通过比较野生型与PtomtAPX-OE植株中可溶性总蛋白含量、相对含水量、叶绿素含量等生理反应的相关指标，发现转基因植株在受到胁迫处理后均升高。这些指标检测结果和之前的研究结果几乎一致[26]。MDA含量明显较低，作为细胞损伤程度的指标丙二醛[27]，在干旱胁迫、盐胁迫和氧化胁迫后，过表达烟草中的增加值明显比野生型少，表明烟草有消除胁迫所造成的损伤的能力。此外更多的AsA消耗量，升高的SOD酶活性，降低的H2O2含量，增大的NADP/NADPH比值，表明PtomtAPX具有H2O2清除能力，这使得细胞损伤较小，在逆境中的耐性更强。
以上结果表明，PtomtAPX-OE提高了烟草的抗逆性，能够有效降解H2O2，对烟草的抗逆具有重要作用。这一结论为PtomtAPX基因在毛白杨中的研究奠定了研究基础，为后续研究提供了方向。
Tobacco overexpression Populus tomentosa mitochondria ascorbate peroxidase improving stress resistance
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摘要:
目的 为了研究毛白杨线粒体APX（PtomtAPX）在抗逆过程中的作用，本研究对过表达PtomtAPX的转基因烟草进行抗逆研究。 方法 对过表达PtomtAPX烟草和野生型烟草进行干旱、盐、氧化胁迫处理后，测量相对含水量、叶绿素含量、丙二醛含量、APX活性、AsA消耗量、NADP/NADPH比值和SOD活性。 结果 通过对比转基因烟草植株与野生型植株的生长差异发现，在氧化胁迫、盐胁迫和干旱胁迫下，转PtomtAPX基因烟草的APX活性、相对含水量、叶绿素含量、AsA消耗量、NADP/NADPH比值升高量均明显高于野生型，其中转PtomtAPX基因烟草的APX活性为野生型的1.77倍，平均相对含水量为野生型的1.15倍，叶绿素含量为野生型的1.6倍，AsA消耗量为野生型的1.11倍，NADP/NADPH值为野生型的1.18倍，表明过表达PtomtAPX转基因植株清除活性氧的能力更强。 结论 在非生物胁迫下，PtomtAPX能消除H2O2，防止细胞损伤，在植物抗逆中发挥重要作用。 Abstract:Objective In order to study the role of Populus tomentosa mitochondrial APX (PtomtAPX), the stress resistance of overexpression PtomtAPX transgenic tobacco was studied in this paper. Method Overexpression PtomtAPX transgenic tobacco and wild type tobacco were treated with drought stress, salt stress and oxidative stress. Relative water content, chlorophyll content, malondialdehyde content, APX activity, AsA consumption, NADP/NADPH ratio, SOD activity were measured. Result In this study, compared wild type plants with overexpression PtomtAPX, under oxidative stress, salt stress and drought stress, the APX activity, relative water content, chlorophyll content, AsA consumption, and NADP/NADPH ratio increase of the tobacco overexpression PtomtAPX gene were significantly higher than those of wild type, of which the APX activity of PtomtAPX transgenic tobacco was 1.77 times of wild type, the average relative water content was 1.15 times of wild type, the chlorophyll content was 1.6 times of wild type, AsA consumption was 1.11 times of wild type, and the ratio of NADP to NADPH was 1.18 times of the wild type. It suggested that the tobacco overexpressed PtomtAPX harbored stronger ROS scavenging ability. Conclusion These indicate that mitochondrial APX acts a crucial role of abiotic stress tolerance in plants by eliminating H2O2 and preventing cell damage under abiotic stress. -
Key words:
- ascorbate peroxidase /
- transgenic tobacco /
- abiotic stress /
- Populus tomentosa
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图 1 PtomtAPX-OE烟草与野生型烟草在干旱环境下的应答反应
CK为对照，II-1 ~ II-3为过表达烟草3个不同的株系。下同。CK is control，II-1 − II-3 represent the three different strains of overexpressed tobaccos. The same below.
Figure 1. Responses of overexpressed APX transgenic tobaccos and wild type under drought environment
图 2 干旱胁迫下野生型与PtomtAPX-OE烟草的差异
Figure 2. Differences between wild type and APX-overexpressed tobaccos under drought stress
图 3 干旱胁迫下PtomtAPX-OE烟草与野生型其他生理应答的差异
Figure 3. Differences in physiological responses of APX-overexpressed tobacco and wild type under drought stress
图 4 盐胁迫对PtomtAPX-OE烟草与野生型烟草叶片的影响
Figure 4. Effects of salt stress on APX-overexpressed tobacco and wild type tobacco leaves
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