过量表达番茄类囊体膜抗坏血酸过氧化物酶基因（StAPX）提高了烟草种苗的抗氧化能力

为了探讨叶绿体类囊体膜抗坏血酸过氧化物酶（tAPX）与其抗氧化性的关系,从番茄叶片中分离了叶绿体类囊体膜抗坏血酸过氧化物酶基因（StA跚并转入到烟草中。以野生型（WT）、转正义StAPX烟草株系T3-3和T3-6为试材,测定了外源过氧化氢诱导的氧化胁迫条件下APX酶活性、过氧化氢酶（CAT）活性、过氧化氢（H2O2）含量、叶绿素荧光参数及叶绿素含量等。Northern杂交显示StAPX因的表达受外源H2O2氧化胁迫的诱导。氧化胁迫下转基因烟草的APX酶活性和清除H2O2的能力都显著高于野生型,并且转基因烟草比野生型具有更高的PSII最大光化学效率及叶绿素含量。结果表明,．刚尸舶勺过量表达有助于提高外源H2O2诱导的转基因烟草的抗氧化能力。     

过表达单脱氢抗坏血酸还原酶基因提高番茄抗UV-B胁迫能力

以野生型（WT）和转正义叶绿体单脱氢抗坏血酸还原酶基因（LeMDAR）番茄为试材,探讨了UV-B胁迫下过表达LeMDAR对番茄抗氧化能力的影响。测定了不同时间uV-B处理下番茄抗坏血酸（AsA）含量,脱氢抗坏血酸（DHA）含量,单脱氢抗坏血酸还原酶（MDAR）活性,光合速率和叶绿素荧光参数等。在UV-B处理下,转基因番茄植株的AsA含量、MDAR酶及抗坏血酸过氧化物酶（APx）活性、H：0：和超氧阴离子清除速率、净光合速率（只）高于野生型番茄。此外,紫外胁迫下,转基因株系丙二醛（MDA）含量和相对电导率（REC）较野生型增加的少。上述结果表明,MDAR对抗抗坏血酸再生具有重要作用,过表达LeMDAR提高了番茄植株抗氧化能力,对光合机构有保护作用。     

过表达ZmSKIP基因提高烟草抗低温胁迫的能力

以野生型和过表达ZmSKIP基因烟草为试材, 研究了低温胁迫下过表达ZmSKIP对烟草抗氧化能力的影响。测定了不同低温处理时间下过表达ZmSKIP转基因烟草T3代植株和野生型植株抗氧化酶如超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）活性和丙二醛（MDA）含量以及相对电导率, 结果表明, 低温下, 相对于野生型植株, 转基因烟草具有较高的抗氧化酶活性和较低的相对电导率和MDA含量, 说明过表达ZmSKIP提高了转基因植株的耐低温胁迫能力。     

过表达LeNLP4转录因子提高番茄抗低温胁迫能力

NAC（NAM-ATAF1,2-CUC2）转录因子在植物胁迫响应中起重要作用。为了探讨三舭丹基因在番茄抗低温胁迫中的功能,分离了番茄LeNLP4转录因子基因,并获得转正义LeNLP4基因番茄植株。荧光定量PCR分析表明,LeNLP4的表达受低温诱导。与野生型植株相比,在4℃胁迫下转基因植株具有较高的生长量和光系统II（PSH）最大光化学效率（Fv／Fm）、过氧化氢（H2O2）和超氧阴离子（O2-）清除速率、抗坏血酸过氧化物酶（APX）和超氧化物歧化酶（SOD）活性,以及较低的丙二醛（MDA）含量和相对电导率（REC）。过表达株系中SICBF1的表达高于野生型。上述结果表明,LeNLP4的过表达提高了转基因番茄抗低温胁迫能力。     

抑制番茄叶绿体DnaJ蛋白基因的表达降低转基因番茄的抗高温能力

DnaJ蛋白是广泛存在于植物细胞内的一种分子伴侣。在胁迫条件下它能够保护细胞内蛋白质等结构的稳定性。本研究克隆到一个番茄叶绿体DnaJ蛋白基（LeCDJI）。半定量PcR分析表明LeCDJ1,的表达受NaCl、高温、PEGTLABA的诱导。利用反义抑制的方法获得了LeCDJl抑制表达的转基因番茄。高温条件下,转基因株系较野生型表现出较严重的光系统II（PsII）光抑制,较低的D1蛋白含量,较高的超氧阴离子（O2-）、过氧化氢（H2O2）含量,及较低的抗坏血酸过氧化物酶（APX）和超氧化物歧化酶（SOD）活性。这些结果表明,抑制LeCDJ1的表达降低了转基因番茄的抗高温能力。     

烟草叶片发育过程中光合功能衰退与H2O2积累的关系

以烟草（Nicotiana tabacum L．cv NC89）为材料,研究了叶片发育过程中H2O2积累与叶绿体光合功能衰退、抗坏血酸-谷胱甘肽（AsA—GSH）循环的关联。结果表明,光合功能衰退过程中,各光合参数均表现为先缓慢后快速的下降趋势,核酮糖-1,5-二磷酸羧化酶（RuBPCase）活性下降较电子传递活性下降迅速,H2O2含量与叶绿素含量、光合速率、RuBPCase活性、抗坏血酸过氧化物酶（APX）、谷胱甘肽还原酶（GR）活性显著负相关。H2O2的定位染色也证实光合功能衰退与H2O2积累密切相关。APX和GR在光合功能可逆衰退阶段维持较高水平,不可逆衰退阶段下降稍快。烟草叶片光合功能衰退快于AsA—GSH循环运转的下调。     

超表达拟南芥2-烯醛还原酶基因对烟草抗旱性的作用机理分析

为研究是否可以利用2-烯醛还原酶(AER)来清除活性氧下游的醛自由基达到提高植物的抗旱性,以超表达拟南芥AER基因烟草和野生型烟草(SR)为研究材料,利用干旱胁迫处理进行抗旱性分析,测定了干旱胁迫及复水后各个烟草株系的生物量、光合速率、叶绿素荧光参数、叶绿素含量、MDA和H2O2含量等指标。结果显示:(1)干旱胁迫下,转基因烟草株系的生物量、叶绿素含量、净光合速率、PSⅡ最大光化学效率及H2O2的清除能力均显著高于对照;(2)复水之后,烟草植株的各项生理指标都得到一定程度的恢复,而转基因株系相比于野生型恢复迅速,恢复能力更强。研究认为,超表达AER基因可以通过清除活性氧及其下游醛自由基来提高烟草的抗旱能力。     

干旱胁迫下CO2浓度升高对转StAPX番茄植株耐旱能力的影响

在干旱胁迫伴随大气CO2浓度以及升高的CO2浓度（加倍）条件下,以过量表达番茄类囊体膜抗坏血酸过氧化物酶基因（StAPX）的转基因番茄为试材,探明干旱胁迫TCO2浓度升高对转基因及其野生型番茄植株清除活性氧及耐旱能力的影响。结果表明：升高的CO2浓度明显增加了干旱胁迫下植物的光合水平;升高的CO2浓度明显降低了干旱导致的植物体内H2O2．和O2的积累,影响了干旱胁迫下番茄植株的水．水循环系统的活性氧清除酶活性和小分子抗氧化物质含量;干旱胁迫下即使伴随升高的CO2浓度,测试番茄植株体内的渗透调节物质含量变化也不太明显;升高的CO2浓度明显降低了干旱胁迫下的植物细胞膜伤害程度;干旱胁迫下,升高的CO2浓度对转基因番茄株系比对野生型植株的影响更加明显。结果证明干旱逆境下,升高的CO2浓度能够在一定程度上进一步提高转基因番茄植株的耐旱性。     

水稻OsMGD2和OsMGD3基因的功能鉴定及对烟草耐低磷胁迫的影响

【目的】单半乳糖甘油二酯合酶（MGD）是合成单半乳糖甘油二酯（MGDG）的关键酶,在植物响应低磷胁迫过程中起着重要作用。为系统了解水稻OsMGD2和OsMGD3基因在低磷胁迫响应中的作用和功能。【方法】采用盆栽试验分析正常和低磷条件下野生型（SR1）和过表达OsMGD2和OsMGD3转基因烟草的生理响应和脂质组分的变化。【结果】无论在正常还是低磷条件下,转基因烟草与野生型的磷含量无显著差异,然而转基因烟草的生物量、叶绿素含量和光合能力均显著高于野生型。转基因烟草在低磷胁迫下的磷脂（PL）含量、双半乳糖甘油二酯（DGDG）含量、DGDG/MGDG比值和半乳糖脂（GL）/PL比值均显著高于野生型烟草,且OsMGD3转基因烟草的脂质含量和比值均高于OsMGD2转基因烟草。【结论】通过调控水稻OsMGD2/3基因表达,可提高植物低磷下的膜脂重塑能力,维持植物在低磷胁迫下的较高的光合和生长能力,增加植物的低磷耐受性。     

抗氧化系统参与循环干旱锻炼提高烟草植株抗旱性的形成

对漂浮育苗的烟草幼苗进行控水-半萎焉-复水-恢复的循环干旱锻炼。结果表明,这种干旱锻炼能显著提高烟草叶片抗氧化剂谷胱甘肽（GSH）、抗坏血酸（AsA）的含量、还原型抗氧化剂在总抗氧化剂中的比例和抗氧化酶包括超氧化物歧化酶（SOD）、抗坏血酸过氧化物酶（APX）、过氧化氢酶（CAT）、愈创木酚过氧化物酶（GPX）、谷胱甘肽还原酶（GR）的活性,降低丙二醛（MDA）的含量。当烟草植株遭受后续的干旱胁迫时,与未锻炼的对照相比,干旱锻炼过的烟草植株能保持较高的GSH和AsA含量、还原型抗氧化剂在总抗氧化剂中的比例和抗氧化酶（SOD、APx、CAT、GPX和GR）活性,以及较低的MDA含量,表明这种循环干旱锻炼提高了细胞抗氧化能力,有助于缓解烟草植株由干旱引起的氧化胁迫及其所导致的伤害,最终提高其抗旱性。这些结果表明。抗氧化系统参与了循环干旱锻炼提高烟草植株抗旱性的形成过程。     

Suppression of tomato <Emphasis Type="Italic">SlGGP</Emphasis> aggravates methyl viologen-mediated oxidative stress

Ascorbate (AsA) is an important antioxidant that can scavenge reactive oxygen species to protect plant cells against oxidative stress. Guanosine 5'-diphosphate (GDP)-L-galactose phosphorylase (GGP) is a key enzyme in the AsA biosynthetic pathway. To investigate the functions of GGP in AsA synthesis and oxidative stress tolerance in tomato, antisense lines with a reduced expression of SlGGP were obtained. Photobleaching after treatment of leaf disks with methyl viologen was more severe in transgenic lines compared to wild type (WT) plants. Moreover, compared with the WT plants, the transgenic plants showed a higher content of hydrogen peroxide, superoxide anion, malondialdehyde, as well as ion leakage, but a lower content of AsA and chlorophylls, ascorbate peroxidase activity, net photosynthetic rate, and maximal photochemical efficiency of photosystem II. Results of real-time quantitative polymerase chain reaction show that suppression of the SlGGP gene in the transgenic plants reduced their oxidative stress tolerance.     

Overexpression of tomato GDP-l-galactose phosphorylase gene in tobacco improves tolerance to chilling stress

Key message

The overexpression of tomato GDP- l -galactose phosphorylase gene enhanced tolerance to chilling stress and reduced photoinhibition of photosystems I and II in transgenic tobacco.

Abstract

Chilling stress is a crucial factor that limits the geographical distribution and yield of chilling-sensitive plants. Ascorbate (AsA) protects plants by scavenging reactive oxygen species and reduces photoinhibition by promoting the conversion of violaxanthin to zeaxanthin in the xanthophyll cycle to dissipate excess excitation energy. Possible mechanisms of AsA for plant photoprotection under chilling stress were investigated by isolating the tomato GDP-l-galactose phosphorylase gene (SlGGP) and producing transgenic tobacco plants with overexpression of SlGGP. The transgenic plants subjected to chilling stress accumulated less H₂O₂, demonstrated lower levels of ion leakage and malondialdehyde, and acquired higher net photosynthetic rate, higher maximum photochemical efficiency of PSII, and higher D1 protein content compared with the wild-type (WT) plants. The transgenic plants subjected to chilling stress also showed higher GDP-l-galactose phosphorylase activity, increased AsA content as well as ascorbate peroxidase and oxidizable P700 activities than WT plants. Thus, SlGGP overexpression is crucial in promoting AsA synthesis and alleviating photoinhibition of two photosystems.     

Overexpression of chloroplastic monodehydroascorbate reductase enhanced tolerance to temperature and methyl viologen‐mediated oxidative stresses

A tomato (Lycopersicon esculentum Mill.) monodehydroascorbate reductase gene (LeMDAR) was isolated. The LeMDAR–green fluorescence protein (GFP) fusion protein was targeted to chloroplast in Arabidopsis mesophyll protoplast. RNA and protein gel blot analyses confirmed that the sense‐ and antisense‐ LeMDAR were integrated into the tomato genome. The MDAR activities and the levels of reduced ascorbate (AsA) were markedly increased in sense transgenic lines and decreased in antisense transgenic lines compared with wild‐type (WT) plants. Under low and high temperature stresses, the sense transgenic plants showed lower level of hydrogen peroxide (H₂O₂), lower thiobarbituric acid reactive substance (TBARS) content, higher net photosynthetic rate (P_n), higher maximal photochemical efficiency of PSII (F_v/F_m) and fresh weight compared with WT plants. The oxidizable P700 decreased more obviously in WT and antisense plants than that in sense plants at chilling temperature under low irradiance. Furthermore, the sense transgenic plants exhibited significantly lower H₂O₂ level, higher ascorbate peroxidase (APX) activity, greater P_n and F_v/F_m under methyl viologen (MV)‐mediated oxidative stresses. These results indicated that overexpression of chloroplastic MDAR played an important role in alleviating photoinhibition of PSI and PSII and enhancing the tolerance to various abiotic stresses by elevating AsA level.     

Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress

To evaluate the physiological importance of thylakoid membrane-bound ascorbate peroxidase (tAPX) in the active oxygen species-scavenging system of chloroplasts, the level of tAPX in tobacco plants was altered by expression of the tAPX cDNA in both sense and antisense orientation. The tobacco plants transformed with constructs of antisense tAPXs from spinach and tobacco could not be obtained, suggesting that the suppression of tAPX in higher plants had a severe effect on the growth even under normal conditions. In contrast, the transgenic tobacco plants (TpTAP-12) overexpressing tAPX, which had approximately 37-fold higher activity than that of the wild-type plants, were generated. The TpTAP-12 plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV, 50 microm) under light intensity (300 and 1600 microE m(-2) sec(-1)) and by chilling stress with high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)). At 24 h after the MV treatment under illumination at 300 microE m-2 sec-1, destruction of chlorophyll was observed in the wild-type plants, but not in the TpTAP-12 plants. The activities of thiol-modulated enzymes in the Calvin cycle, the level and redox status of ascorbate (AsA), and the activity of tAPX in the wild-type plants significantly decreased, while those in the TpTAP-12 plants were hardly changed. These observations suggest that tAPX is a limiting factor of antioxidative systems under photo-oxidative stress in chloroplasts, and that the enhanced activity of tAPX functions to maintain the AsA content and the redox status of AsA under stress conditions.     

Overexpression in tobacco of a tomato GMPase gene improves tolerance to both low and high temperature stress by enhancing antioxidation capacity

GDP-mannose pyrophosphorylase (GMPase: EC 2.7.7.22) plays a crucial role in the synthesis of l-ascorbate (AsA) and the consequent detoxification of reactive oxygen species (ROS). Herein, a GMPase (accession ID DQ449030) was identified and cloned from tomato. The full-length cDNA sequence of this gene contains 1,498 bp nucleotides encoding a putative protein with 361 amino acid residues of approximate molecular weight 43 kDa. Northern blot analysis revealed that the GMPase was expressed in all examined tomato tissues, but its expression level was up-regulated in tomato plants subjected to abnormal temperatures. We then overexpressed this tomato GMPase in tobacco plants and observed that the activity of GMPase and the content of AsA were significantly increased by two- to fourfold in the leaves of transgenic tobacco plants. The effect of this gene overexpression was superimposed by the treatments of high or low temperature in tobacco, since the activities of both chloroplastic SOD (superoxide dismutase EC 1.15.1.1), APX (ascorbate peroxidase EC 1.11.1.7) and the content of AsA in leaves were significantly higher in transgenic plants than those of WT, while the contents of H₂O₂ and O₂ ^−· were reduced. Meanwhile, relative electric conductivity increased less in transgenic plants than that in WT, and the net photosynthetic rate (P _n) and the maximal photochemical efficiency of PSII (F _v/F _m) of transgenic plants were notably higher than those of WT under temperature stresses. In conclusion, the overexpression of GMPase increased the content of AsA, thereby leading to the increase in tolerance to temperature stress in transgenic plants.     

盐胁迫下过量表达腺苷甲硫氨酸合成酶基因的转基因烟草的生长

以转腺苷甲硫氨酸合成酶基因（SsSAMS2）烟草纯合子ST8-9为实验材料,研究盐胁迫下过量表达SsSAMS2对转基因烟草生长影响的结果表明,200mmol·L-1NaCl处理后的转基因烟草的光合速率和生物量都比野生型烟草高,积累的自由多聚胺比较多,同时精氨酸脱羧酶的转录本也更丰富。显示转基因烟草的耐盐性比野生型烟草高,多聚胺在烟株缓解盐胁中可能是起了重要作用。