榆钱菠菜脯氨酸转运蛋白基因的克隆及转基因拟南芥的耐盐性

脯氨酸是自然界中分布最广泛,作用最重要的渗透保护剂之一,同时又是高等植物中一类重要的碳源和氮源物质.为了解环境胁迫下脯氨酸的转运调节,从一个典型的盐生植物榆钱菠菜( Atriplex hortensis L.)中通过cDNA文库筛选和5′-RACE的方法获得了一个全长的cDNA (AhProT1),其编码蛋白与脯氨酸转运蛋白有60%～69%的同源性,含有11个跨膜结构域.聚类分析表明,微生物和高等植物的脯氨酸转运蛋白同源程度相对高于哺乳动物.为进一步分析脯氨酸转运蛋白在植物中的功能,将AhProT1置于35S启动子下转入拟南芥(Arabidopsis thaliana).通过同位素示踪法发现, 与对照植物相比, 转基因植物在根中积累更多的脯氨酸;在一系列不同浓度的盐胁迫试验中,转基因植株最高可耐受200 mmol/L NaCl,并可持续生长,而对照植株在150 mmol/L NaCl下即已死亡.     

榆钱菠菜脯氨酸转运蛋白基因的克隆及转基因拟南芥的耐盐性

脯氨酸是自然界中分布最广泛 ,作用最重要的渗透保护剂之一 ,同时又是高等植物中一类重要的碳源和氮源物质。为了解环境胁迫下脯氨酸的转运调节 ,从一个典型的盐生植物榆钱菠菜 (AtriplexhortensisL .)中通过cDNA文库筛选和 5′_RACE的方法获得了一个全长的cDNA (AhProT1) ,其编码蛋白与脯氨酸转运蛋白有 6 0 %～ 6 9%的同源性 ,含有 11个跨膜结构域。聚类分析表明 ,微生物和高等植物的脯氨酸转运蛋白同源程度相对高于哺乳动物。为进一步分析脯氨酸转运蛋白在植物中的功能 ,将AhProT1置于 35S启动子下转入拟南芥 (Arabidopsisthaliana)。通过同位素示踪法发现 ,与对照植物相比 ,转基因植物在根中积累更多的脯氨酸 ;在一系列不同浓度的盐胁迫试验中 ,转基因植株最高可耐受 2 0 0mmol/LNaCl,并可持续生长 ,而对照植株在 15 0mmol/LNaCl下即已死亡。     

Overexpression of Arabidopsis Molybdenum Cofactor Sulfurase Gene Confers Drought Tolerance in Maize (Zea mays L.)

Abscisic acid (ABA) is a key component of the signaling system that integrates plant adaptive responses to abiotic stress. Overexpression of Arabidopsis molybdenum cofactor sulfurase gene (LOS5) in maize markedly enhanced the expression of ZmAO and aldehyde oxidase (AO) activity, leading to ABA accumulation and increased drought tolerance. Transgenic maize (Zea mays L.) exhibited the expected reductions in stomatal aperture, which led to decreased water loss and maintenance of higher relative water content (RWC) and leaf water potential. Also, transgenic maize subjected to drought treatment exhibited lower leaf wilting, electrolyte leakage, malondialdehyde (MDA) and H₂O₂ content, and higher activities of antioxidative enzymes and proline content compared to wild-type (WT) maize. Moreover, overexpression of LOS5 enhanced the expression of stress-regulated genes such as Rad 17, NCED1, CAT1, and ZmP5CS1 under drought stress conditions, and increased root system development and biomass yield after re-watering. The increased drought tolerance in transgenic plants was associated with ABA accumulation via activated AO and expression of stress-related gene via ABA induction, which sequentially induced a set of favorable stress-related physiological and biochemical responses.     

过表达海洋微生物宏基因组MbCSP提高转基因拟南芥的抗旱和耐寒性

干旱和低温是影响农作物生长发育的重要因素,培育转基因作物是解决此问题的有效途径。冷激蛋白（Cold Shock Proteins,CSPs）是一类高度保守的核酸结合蛋白,参与非生物胁迫应答等细胞生理活动,转CSP基因可增强作物抗逆能力。以海洋微生物宏基因组DNA为模板,采用锚定PCR的方法克隆得到了MbCSP基因,其ORF为216 bp,编码一个由71个氨基酸构成的蛋白;对其进行同源性分析,显示该氨基酸序列与EcCSPG、EcCSPA（大肠杆菌 Escherichia coli）,BsCspB（枯草芽孢杆菌Bacillus subtilis）和BcCspA（蜡样芽孢杆菌 Bacillus cereus）等冷休克蛋白氨基酸序列同源性在60%~90%;对该氨基酸序列进行多重序列比对和系统发育树分析,结果发现MbCSP蛋白包含RNP1（KGFGFI）和RNP2（VFVHF）等CSP蛋白经典的保守结构域,其与EcCspG（大肠杆菌）和CmCspG、CmCspB（堆肥宏基因组）等生物的冷休克蛋白亲缘关系较近。为进一步探讨冷休克蛋白MbCSP的功能,构建了植物表达载体pTF101-MbCSP,采用花序浸染法转化拟南芥,经过除草剂筛选和PCR检测,获得转基因植株。进行半定量RT-PCR分析,选择表达量最高的阳性株系进行后续的生理检测。结果表明：在干旱胁迫及低温胁迫下,转基因拟南芥的生长状况明显优于野生型,其生物量显著高于野生型植株;转基因拟南芥的叶片相对含水量、叶绿素含量和超氧化物歧化酶活性均高于野生型拟南芥,而丙二醛含量则低于野生型拟南芥。上述结果表明,过表达海洋微生物宏基因组MbCSP能够提高转基因拟南芥的抗旱和耐寒能力,为培育转基因作物新品种奠定了基础。     

Overexpression of SoACLA-1 Gene Confers Drought Tolerance Improvement in Sugarcane

Drought is one of the most severe stresses which limit sugarcane production in China. ATP citrate lyase (ACL) is a major enzyme responsible for the production of acetyl-CoA in cytoplasm and plays an important role in plant metabolism and stress response. In this study, sugarcane ACL gene SoACLA-1 was cloned. The plant overexpression vector of SoACLA-1 was built and transformed into sugarcane calli by Agrobacterium-mediated transformation, and PCR analysis confirmed that SoACLA-1 gene had been stably present in the T0, T1, and T2 generations of the transgenic sugarcane. In order to evaluate the drought resistance of the transgenic lines and verify the function of SoACLA-1 gene in the transgenic sugarcane, T1 generation of the SoACLA-1 transgenic sugarcane lines was used as the material to investigate the physiological and biochemical characteristics at 0 day, 3 days, 6 days, and 9 days after water stress and rewatering for 3 days. Comprehensive evaluation of four indicators (chlorophyll, malondialdehyde, proline, soluble sugar) related to drought resistance was done with membership fuzzy function method. The results showed that the drought resistance of five transgenic sugarcane lines from strong to weak, in turn, was RT2?>?RT4?>?RT3?>?RT1?>?WT, and the recovery ability after drought, in turn, was RT1?>?RT2?>?RT4?>?RT3?>?WT. The T2 generation of the SoACLA-1 transgenic sugarcane lines was used to analyze the physiological and biochemical changes and the expression of drought-related genes under water stress. The results showed that the transgenic sugarcane lines were more tolerant to drought as compared with the wild-type plants. Our findings indicated that SoACLA-1 gene plays an important role as a positive factor in response to water stress, and overexpression of SoACLA-1 can enhance drought tolerance in transgenic sugarcane plants.

     

过量表达棉花CBF2基因提高转基因拟南芥抗旱耐盐能力

CBF/DREB是一类植物中特有的转录因子,在植物抵抗逆境胁迫过程中发挥重要功能。本研究从陆地棉(Gossypium hirsutum L.)Coker 312中克隆获得1个棉花CBF/DREB基因,命名为Gh CBF2,该基因编码一个由216个氨基酸组成的CBF蛋白。序列分析结果显示,Gh CBF2与其他植物的CBF蛋白类似,含有AP2转录因子典型的保守结构域。干旱或高盐胁迫处理明显增加了Gh CBF2基因的表达量。亚细胞定位分析结果发现Gh CBF2定位在细胞核中。将Gh CBF2基因构建到由35S启动子调控的植物表达载体p MD上并转化拟南芥(Arabidopsis thaliana L.),结果表明,在干旱和盐胁迫条件下,过量表达Gh CBF2基因拟南芥的成活率显著高于野生型,并且游离脯氨酸和可溶性糖含量也高于野生型,说明转Gh CBF2基因提高了拟南芥的耐盐抗旱能力。采用实时荧光定量PCR方法分析胁迫相关标记基因COR15A、RD29A和ERD6的表达情况,结果显示转基因株系中的表达量显著高于野生型,说明Gh CBF2参与调控拟南芥干旱和盐胁迫相关基因的表达。     

过表达IbOr基因甘薯增强抗旱性的生理机制

以超表达甘薯橙色基因(IbOr)的转基因甘薯(TS)以及非转基因甘薯(NT)为实验材料,通过15%聚乙二醇6000(PEG-6000)模拟干旱条件,研究转基因与非转基因甘薯幼苗在水分胁迫不同时间的光合系统,膜脂过氧化及抗氧化防御系统中主要指标的变化情况,探讨转基因甘薯耐旱性的生理机制。结果显示:(1)随PEG-6000胁迫时间延长,甘薯叶片的叶绿素、类胡萝卜素含量及其叶片净光合速率、气孔导度、胞间CO2浓度、蒸腾速率都显著降低,但转基因株系降低幅度小于非转基因植株。(2)在正常供水和水分胁迫下,超表达IbOr基因甘薯叶片中O-·2、MDA含量均低于非转基因甘薯,即转基因甘薯具有较低的活性氧水平且脂膜受损伤较小。(3)PEG-6000胁迫24h后,甘薯叶片中SOD、POD酶活性均增加,48h达到最大值,且转基因甘薯中2种酶活性显著高于非转基因甘薯。研究表明,过表达IbOr基因可以有效减轻甘薯在水分胁迫条件下受损害的程度,且可能主要通过提高甘薯的抗氧化胁迫能力来完成。     

Overexpression of LmgshF from Listeria monocytogenes in Indica Rice Confers Salt Stress Tolerance

Russian Journal of Plant Physiology - Glutathione (γ-glutamylcysteinylglycine; GSH), is a multi-functional tri-peptide antioxidant, a key agent in defense against abiotic and biotic stress. A...     

异源过表达OsATG8b基因提高转基因拟南芥的 氮/碳胁迫耐受性和产量

氮素是参与植物生长发育的一种重要元素, 对植物的产量和品质具有重要作用。自噬是真核生物中一种保守的细胞组分降解-循环再利用途径, 在植物生长发育和籽粒形成期间的氮素再动员过程中发挥作用。我们鉴定到水稻(Oryza sativa)自噬核心基因OsATG8b, 并获得2个独立的35S-OsATG8b转基因拟南芥(Arabidopsis thaliana)纯合株系。研究表明OsATG8b基因响应低氮胁迫处理, 过表达OsATG8b基因促进转基因拟南芥的生长发育, 使莲座叶增大, 单株产量显著提高(15.16%)。进一步研究表明, 过表达OsATG8b能够显著增强缺氮胁迫下转基因拟南芥叶片中的自噬活性, 从而有效缓解氮胁迫和碳胁迫对转基因拟南芥造成的生长抑制。因此, OsATG8b是提高氮素利用效率和产量的候选基因。     

An Acer palmatum R2R3-MYB Gene,ApMYB77, Confers Freezing and Drought Tolerance in Arabidopsis thaliana

Journal of Plant Growth Regulation - Low temperature is one of the most prominent environmental factors affecting plant growth. As a deciduous arboreal tree, Acer palmatum has considerable...     

异源表达CkLEA1基因增强了拟南芥对非生物胁迫的耐受性

植物在生长过程中会受到各种非生物胁迫的伤害,导致生长发育和产量受到严重影响,胚胎晚期丰富蛋白（late embryogenesis abundant proteins,LEA蛋白）在植物抵抗非生物胁迫过程中起着重要的保护作用。在前期的研究基础上,将受多种胁迫诱导的柠条锦鸡儿CkLEA1（GenBank登录号KC309408）基因转入野生型拟南芥,通过实时荧光定量PCR从7株T₃代纯合体中筛选出3个转基因株系做进一步研究。种子萌发率实验发现,在200 mmol/L NaCl和400 mmol/L甘露醇处理下,转基因株系萌发率均高于野生型拟南芥。干旱处理2周大的幼苗后,转基因株系明显比野生型更抗旱,存活率高于野生型,并且失水率低于野生型。同时,转基因株系积累了较少的丙二醛（MDA）,超氧化物歧化酶（SOD）活性和谷胱甘肽（GSH）含量也高于野生型。这些结果表明,柠条锦鸡儿CkLEA1基因在种子萌发阶段提高了拟南芥对盐和渗透胁迫的耐受性,并且提高了转基因拟南芥幼苗生长阶段对干旱胁迫的抵抗能力。     

CaPUB1, a Hot Pepper U-box E3 Ubiquitin Ligase,Confers Enhanced Cold Stress Tolerance and Decreased Drought Stress Tolerance in Transgenic Rice (Oryza sativa L.)

Abiotic stresses such as drought and low temperature critically restrict plant growth, reproduction, and productivity. Higher plants have developed various defense strategies against these unfavorable conditions. CaPUB1 (Capsicum annuum Putative U-box protein 1) is a hot pepper U-box E3 Ub ligase. Transgenic Arabidopsis plants that constitutively expressed CaPUB1 exhibited drought-sensitive phenotypes, suggesting that it functions as a negative regulator of the drought stress response. In this study, CaPUB1 was over-expressed in rice (Oryza sativa L.), and the phenotypic properties of transgenic rice plants were examined in terms of their drought and cold stress tolerance. Ubi:CaPUB1 T3 transgenic rice plants displayed phenotypes hypersensitive to dehydration, suggesting that its role in the negative regulation of drought stress response is conserved in dicot Arabidopsis and monocot rice plants. In contrast, Ubi:CaPUB1 progeny exhibited phenotypes markedly tolerant to prolonged low temperature (4°C) treatment, compared to those of wild-type plants, as determined by survival rates, electrolyte leakage, and total chlorophyll content. Cold stress-induced marker genes, including DREB1A, DREB1B, DREB1C, and Cytochrome P450, were more up-regulated by cold treatment in Ubi:CaPUB1 plants than in wild-type plants. These results suggest that CaPUB1 serves as both a negative regulator of the drought stress response and a positive regulator of the cold stress response in transgenic rice plants. This raises the possibility that CaPUB1 participates in the cross-talk between drought and low-temperature signaling pathways.     

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。