转甜菜碱醛脱氢酶基因马铃薯的抗旱耐盐性

通过根癌农杆菌介导法将甜菜碱醛脱氢酶(BADH)基因导入马铃薯栽培品种甘农薯2号, 经PCR、Southern杂交和Northern杂交证明BADH基因已整合到马铃薯基因组中并在转基因植株中转录和表达。测定表明对照植株没有BADH酶活性, 各转化株系在胁迫前后BADH酶活性近似, 在2~11 U之间。BADH酶活性与叶片的相对电导率呈一定的负相关(y= –3.7738x+57.083, r=0.989^**)。在NaCl和PEG胁迫下, 转基因植株生长正常, 株高比对照提高0.41~1.00 cm, 单株重量比对照增加10%~35%, 说明外源BADH基因的导入提高了马铃薯植株对干旱和盐碱的抗性。     

转甜菜碱醛脱氢酶基因马铃薯的抗旱耐盐性

通过根癌农杆菌介导法将甜菜碱醛脱氢酶(BADH)基因导入马铃薯栽培品种甘农薯2号, 经PCR、Southern杂交和Northern杂交证明BADH基因已整合到马铃薯基因组中并在转基因植株中转录和表达。测定表明对照植株没有BADH酶活性, 各转化株系在胁迫前后BADH酶活性近似, 在2~11 U之间。BADH酶活性与叶片的相对电导率呈一定的负相关(y= –3.7738x+57.083, r=0.989^**)。在NaCl和PEG胁迫下, 转基因植株生长正常, 株高比对照提高0.41~1.00 cm, 单株重量比对照增加10%~35%, 说明外源BADH基因的导入提高了马铃薯植株对干旱和盐碱的抗性。     

Enhancement of drought resistance and biomass by increasing the amount of glycine betaine in wheat seedlings

Drought is a major agricultural menace reducing crop productivity and limiting the successful realization of land potential throughout the world. Therefore, breeding common wheat with improved drought-tolerance via genetic manipulation is of great importance. We have introduced the betA gene encoding choline dehydrogenase from Escherichia coli into common wheat (Triticum aestivum L.) by Agrobacterium-mediated transformation. Various levels of expression of the betA gene were confirmed by RT-PCR among the transgenic lines and different levels of glycine betaine accumulation were detected in these lines. Several wheat transgenic lines with different betA expression levels in the T3 generation and wild-type (WT) were selected to test their performance under drought stress conditions. Water deficit in plants caused a reduction in photosynthesis and activity of the PSII complex and resulted in increased accumulation of osmolytes. Drought stress also led to lower membrane stability along with much higher activities of superoxide dismutase and peroxidase in all wheat lines. However, wheat lines that were transgenic for the betA gene were less injured and exhibited greater root length and growth compared with the WT. It was concluded that the amount of injury to the wheat plants was negatively correlated with the level of accumulation of glycine betaine, and the glycine betaine acted as an important osmoprotectant in transgenic plants to improve root growth, and enhance the resistance of transgenic plants to drought stress.     

转甜菜碱醛脱氢酶基因提高烟草抗旱及耐盐性

将甜菜碱醛脱氢酶（BADH）基因与组成型启动子CaMV 35S启动子融合,构建了植物表达质粒pBIBB。通过根癌农杆菌介导将BADH基因导入烟草,经PCR、Southern杂交、Northern杂交证明BADH基因已整合到烟草基因组中并在转基因植株中转录和表达。测定转基因植株叶片中甜菜碱醛脱氢酶活性,结果显示对照植株没有BADH酶活性,转基因植株的各个株系间甜菜碱醛脱氢酶比活力差异较大,范围在0.1~1.0 U mg-1间。转BADH基因的烟草在盐胁迫和聚乙二醇（PEG）胁迫条件下生长状态良好,生长势强于未转基因植株,说明BADH基因能在异源植物中正常翻译、表达和用于植物抗旱、耐盐基因工程的研究。     

转GsPPCK1基因苜蓿植株的获得及其耐碱性分析

磷酸烯醇式丙酮酸羧化酶激酶(phosphoenolpyruvate carboxylase kinase, PPCK)是一种钙不依赖的丝氨酸/苏氨酸(Ser/Thr)类蛋白激酶, 参与碳氮代谢等多个生物学过程, 然而其在碱胁迫反应中的作用尚未见报道。本研究在前期野生大豆碱胁迫基因表达谱数据基础上, 采用同源克隆的方法分离野生大豆(Glycine soja)PPCK1基因, 该基因与大豆(Glycine max) PPCK1基因(AY374445)具有99%的相似性, 被命名为GsPPCK1。在50 mmol L^–1 NaHCO₃ 胁迫处理3 h内, 根和叶中GsPPCK1基因上调表达, 属碱胁迫早期应答基因。通过农杆菌介导法对肇东苜蓿进行遗传转化, 并对RT-PCR阳性的超表达转基因株系进行耐碱性分析表明, 在100 mmol L^–1 NaHCO₃处理15 d后转基因株系生长状态良好, 而非转基因对照株系明显萎蔫、失绿、甚至死亡; 转基因株系的丙二醛含量和相对质膜透性显著低于非转基因株系(P<0.05), 而叶绿素含量和根系活力显著高于非转基因对照(P<0.05), 说明超量表达GsPPCK1基因增强了苜蓿的耐碱能力。以上结果表明, GsPPCK1参于植物耐碱胁迫反应过程, 在碱胁迫基因工程研究领域具有良好的理论和实际应用意义。     

Transformation of tobacco with an Arabidopsis thaliana gene involved in trehalose biosynthesis increases tolerance to several abiotic stresses

Summary Trehalose (a non-reducing disaccharide) plays an important role in abiotic stress protection. It has been shown that using trehalose synthesis genes of bacterial origin, drought and salt tolerance could be achieved in several plants. A cassette harboring the AtTPS1 gene under the control of the CaMV35S promoter and the Bialaphos resistance gene was inserted in the binary plasmid vector pGreen0229 and used for Agrobacterium-mediated transformation of tobacco (Nicotiana tabacum). T0 plants obtained were analyzed by PCR for the presence of AtTPS1 gene. Thirty lines were positive and seeds were germinated on media with 6 mg/l PPT to obtain T1 plants that were grown in the greenhouse to obtain T2 seeds that were germinated on selective media. Lines which seeds showed a 100 % survival rate were considered homozygous transgenic T1 lines. Three lines were selected and gene expression confirmed by northern and western blots. Transgenic seeds were germinated on media with different concentrations of mannitol (0, 0.25, 0.5 and 0.75 M) and sodium chloride (0, 0.07, 0.14, 0.2, 0.27 and 0.34 M) to score their tolerance to osmotic stress. Assays were conducted to test the tolerance of transgenic plants to drought (measurement of water percentage as a consequence of water withdrawal), desiccation (measurement of water loss as a consequence leaf detaching) and temperature stresses (germination at 15 ^∘C and 35^∘C). Transgenic tobacco plant lines registered higher germination rates under osmotic and temperature stress situations than did wild-type plants. Responses to drought and desiccation stresses were similar for all plant lines. It can hence be suggested that the heterologous expression of TPS1 gene from Arabidopsis can be used successfully to increase abiotic stress tolerance in model plants and probably in other crops.     

Morphological and biochemical responses of Balanites aegyptiaca to drought stress and recovery are provenance‐dependent

Balanites aegyptiaca is a drought‐tolerant tree naturally distributed in Africa and has a high potential for biofuel production and livelihood. To understand the plant tolerance to drought stress, B. aegyptiaca plants collected from five provenances were subjected for 4 weeks to drought stress through different regimes of soil volumetric water content (VWC, i.e. 25% control, 15% as moderate and 5% as a severe drought stress) followed by 2‐week recovery. Morpho‐physiological responses as well as the changes in antioxidant defences under water stress and recovery were investigated. Drought stress significantly reduced plant biomass‐related parameters, stomatal conductance, quantum efficiency and increased leaf temperature. Each provenance showed specific patterns of stress response reactions that were detected in a cluster analysis. The large leaf area and a high level of lipid peroxidation in Cairo provenance increased its sensitivity to severe drought. For provenances El‐Kharga and Yemen, the highest tocopherol contents and the highest catalytic activities of ascorbate peroxidase (SOD), catalase (CAT), glutathione reductase (GR) and dehydroascorbate reductase (DHAR) were recorded. These traits contributed to the high drought tolerance of these two provenances in comparison with the other provenances. All plants recovered from stress and showed specifically increased activity of glutathione‐S‐transferase (GST) as a repair mechanism. Results showed that the drought tolerance level in B. aegyptiaca is provenance‐dependent.     

转ppc基因水稻苗期抗旱特性研究

通过C₄转基因技术改善C₃作物光合作用, 以期提高作物产量是国内外研究的热点之一。然而, 目前关于转磷酸烯醇式丙酮酸羧化酶(PEPC)基因对水稻的光合作用、产量和抗旱性的影响及其调节机理仍不很清楚。本研究以T₄代转ppc基因水稻为材料, 进行产量和苗期抗旱性研究。结果表明, 在旱作栽培条件下, 两个转ppc基因株系(T1, T2)单株产量分别比未转基因的对照(WT)增产28%和42%, 分蘖增加27%和40%; T1和T2可维持较高的光合速率、气孔导度和水分利用效率; 干旱胁迫使超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量增加, T1和T2 SOD活性增幅(+25%)都显著高于WT(+9%), 而MDA含量增幅显著低于WT, 表明转ppc基因水稻具有较强的抗氧化能力; T1和T2比WT具有较高的脯氨酸含量和渗透势下降幅度, 说明转PEPC基因水稻的渗透调节能力高于对照。PEG-6000处理下, 得到的结果相似。     

Expression analysis of the genes involved in osmotic adjustment in bread wheat (Triticum aestivum L.) cultivars under terminal drought stress conditions

Osmotic adjustment is the main component for physiological machinery of wheat drought tolerance. Some of parameters implicated in osmotic adjustment in 15 bread wheat cultivars were evaluated at soil water deficits (50% FC) and FC as the control in the greenhouse. For the physiological traits, analysis of variance showed that there are highly significant differences between treatments, i.e. water stress levels and wheat cultivars. Shahpasand and Marvdasht as sensitive wheat cultivars had significantly lower osmotic adjustment, relative water content, K+ content, soluble sugar, proline, and glycine betaine levels than the rest of the cultivars. The results of cluster analysis revealed that all cultivars were grouped into three distinct clusters. Dez, Kavir, Pishtaz, and Maron cultivars which have the highest osmoregulation activity were in cluster I, whereas clusters II (Pishgam, Aflak, Hirmand, Zagros, and Vee/Nac) and III (Ws-89-2, Sardari, Azar2, Shapasand, and Marvdasht) had intermediate activity and the lowest capacities for osmoregulation, respectively. In addition, in wheat flag leaf during the reproductive stage under drought conditions, the changes in gene expression of two key genes namely P5CS (D-Pyrroline-5-carboxylate synthetase) and BADH (Betaine Aldehyde Dehydrogenase) in two selected cultivars including Dez and Marvdasht revealed that water stress can increase the expression level of the genes P5CS and BADH in the resistant cultivar, Dez, compared with Marvdasht, the sensitive one. In general, it seemed that application of the all cultivars in cluster I would enable breeders to acquire more reliable achievements under drought conditions.     

变水处理条件下小麦幼苗的甜菜碱代谢与抗旱性的关系

在缓慢土壤水分胁迫一复水的变水处理条件下,小麦幼苗地上部分相对含水量在80%~70%时,甜菜碱含量及甜菜碱醛脱氢酶活性最高,相对含水量大于80%或小于70%,甜菜碱的含量和甜菜碱醛脱氢酶活性都降低,并且两者变化是“同步”的。水分胁迫时,小麦幼苗迅速积累甜菜碱,抗旱型小麦增加6~8倍,水分敏感型增加4倍。积累甜菜碱与     

转MvNHX1和MvP5CS基因棉花耐盐抗旱性比较与育种价值分析

通过对花粉管通道法获得的T7转MvNHX1基因的10个棉花株系和转MvP5CS基因的3个棉花株系与对照非转基因棉花D5在温室内盐和干旱胁迫下的发芽率、生理生化指标以及田间花期干旱胁迫下农艺性状和纤维品质进行分析发现:温室内盐和干旱胁迫后,转基因棉花叶片叶绿素含量、脯氨酸含量均高于对照,而丙二醛含量低于对照;田间花期干旱胁迫下,2种转基因植株果枝数、有效果枝数、铃数、有效铃数、铃重、子棉、皮棉、衣分、子指和衣指均高于对照,说明转MvNHX1基因和转MvP5CS基因植株在干旱逆境下的产量高于非转基因;经花期干旱胁迫后,2种转基因棉花的纤维断裂伸长率、短纤维率、马克隆值和纺纱一致性指数优于非转基因棉花D5。综合分析表明:2种转基因棉花的耐盐抗旱性均有提高,其中转MvNHX1基因棉花的耐盐性优于转MvP5CS基因棉花植株,而转MvP5CS基因棉花植株的抗旱性优于转MvNHX1基因棉花植株。     

转GHABF4转录因子棉花植株的耐盐性研究

AREB/ABFs转录因子家族基因主要参与干旱、高盐、低温等胁迫应答反应。为了获得具有较高耐盐水平的棉花新种质材料,通过农杆菌介导法将耐盐转录因子基因(GHABF4)导入陆地棉中棉35中,通过对转化植株的卡那霉素初步筛选及T1、T2、T3目的基因PCR的分子检测,获得T3转基因棉花纯合系。通过盐胁迫试验对5个T3转基因棉花株系和非转基因棉花对照进行耐盐性分析。结果表明,在200 mmol/L Na Cl胁迫下,与非转基因对照相比,5个转基因棉花株系株高提高2.5~4.4 cm,地上部分的鲜质量增加3.6%~11.8%;且抗氧化物酶SOD、POD、CAT活性以及叶绿素含量提高。在盐胁迫条件下,转GHABF4基因棉花表现出优良的生长和生理优势,转GHABF4基因能够提高棉花的抗盐能力。     

Evaluation and development of flood‐tolerant soybean cultivars

Soybean (Glycine max [L.] Merr.) cultivars are generally sensitive to flooding stress. The plant growth is severely affected and grain yield is largely reduced in the flooded field. It is important to develop flood‐tolerant soybean cultivars for grain production in regions of heavy rainfalls worldwide. In this study, a total of 722 soybean genotypes were evaluated for flooding tolerance at R1 stages (first flower at any node) in the 5‐year flooding screening tests. Differential soybean genotypes exhibited diverse responses to flooding stress with that plant foliar damage score (FDS) and plant survival rate (PSR) ranged from 1.9 to 8.8 and 3.4% to 81.7%, respectively (p < .0001). Based on our standard of flooding evaluation, most genotypes were sensitive to flooding with 6.0 of average FDS and 38.7% of PSR. Fifty‐two soybean genotypes showed flooding tolerance and 11 genotypes were with consistent flooding tolerance during 4‐ to 5‐year continual evaluations. In the meantime, six genotypes were identified with consistent high sensitivity to flooding. The group analysis showed that genotypes from different sources had distinguishable responses to flooding stress (p < .0001). The interacting analysis of year and flooding tolerance indicated that FDS and PSR means were significantly different among 5 years due to weather temperature and flooding treatment time influences of each year (p < .0001). Furthermore, five breeding lines with high‐yielding and flood‐tolerant traits were developed using selected consistent flood‐tolerant and high‐yielding genotypes through conventional breeding approach.     

转BADH基因玉米植株的获得及其耐盐性分析

采用超声波辅助花粉介导植物转基因方法, 将甜菜碱醛脱氢酶(BADH)基因导入玉米自交系郑58, 获得了耐盐性强的转基因玉米植株。经卡那霉素抗性初筛、PCR扩增、Southern blot杂交分析, 证明BADH基因已导入转化植株并整合到其基因组中。用不同浓度的NaCl溶液对T₂代转基因玉米植株与对照进行盐胁迫处理, 结果表明, 转BADH基因玉米植株表现出一定的抗逆性, 生长状况明显优于对照; 根据非转化苗对NaCl的反应以及生长状况, 确定250 mmol L^-1 NaCl溶液为玉米幼苗耐盐性筛选的适宜浓度; 依据此临界浓度下形态指标和生理生化指标的测定结果, 与对照相比, 转基因植株的株高提高10.94%~25.7%, 鲜重增加8.62%~18.2%, 干重增加9%~18.18%, 相对电导率降低37.21%~58.14%, 叶绿素含量增加15.89%~90.65%, 超氧化物歧化酶(SOD)活性提高64.92%~148.29%, 丙二醛(MDA)含量减少26.97%~48.05%。综上所述, 转入甜菜碱醛脱氢酶(BADH)基因提高了玉米的耐盐性。这是首例将BADH基因导入优良玉米自交系郑58的报道。超声波辅助花粉介导法是一种经济、高效、实用和无基因型依赖性的植物基因转化方法。     

Methyl Jasmonate‐Induced Alteration in Lipid Peroxidation,Antioxidative Defence System and Yield in Soybean Under Drought

Methyl jasmonate (MeJA), a plant‐signalling molecule, is involved in an array of plant development and the defence responses. This study was conducted to explore the role of exogenous MeJA application in alleviating the adversities of drought stress in soybean (Glycine max L. Merrill.). Soybean plants were grown under normal conditions until blooming and were then subjected to drought by withholding irrigation followed by foliar application of (50 μm ) MeJA. Drought stress substantially suppressed the yield and yield‐related traits, whereas it accelerated the membrane lipid peroxidation. Nonetheless, substantial increase in activities of enzymatic antioxidants (superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)), proline, relative water contents (RWC) with simultaneous decrease in membrane lipid peroxidation was observed in MeJA‐treated plants under drought. These beneficial effects led to improvement in biological and grain yield, and harvest index under drought. Interestingly, MeJA application was also useful under well‐watered conditions. These results suggest the involvement of MeJA in improving the drought tolerance of soybean by modulating the membrane lipid peroxidation and antioxidant activities.     

转水稻OsSIK1基因玉米植株的获得及抗旱性分析

类受体激酶基因Os SIK1具有通过激活抗氧化系统,增强水稻对于干旱和盐胁迫抗性的作用。为了丰富可利用的作物抗旱基因,获得具有较高抗旱水平的玉米新种质,通过超声波辅助花粉介导法,将水稻类受体激酶基因Os SIK1导入玉米自交系郑58中,并对转化株进行卡那霉素筛选及T1、T2、T3的PCR及Southern Blotting杂交等分子检测,获得转化植株并在T3获得转基因纯合株系。对T3转基因玉米和非转基因玉米对照以16.1%的PEG模拟水分胁迫进行抗旱性分析。结果表明,与对照相比,在水分胁迫处理下,转基因玉米株系叶片相对含水量提高了7.4%~19.8%,叶绿素含量提高了11.3%~106.9%,SOD活性上升45.8%~93.4%,而转基因玉米叶片的相对电导率下降了35.4%~58.1%,MDA含量下降了25.7%~50.4%,说明转Os SIK1基因玉米植株抗旱性得到提高,其中,5个转化株系与对照在抗旱性方面有显著差异,且生长状况明显优于对照。综上所述,研究最终获得5个转Os SIK1基因玉米株系,并证明导入水稻Os SIK1基因可以提高玉米植株的抗旱性。     

Responses to water withdrawal of tobacco plants genetically engineered with the AtTPS1 gene: a special reference to photosynthetic parameters

We have previously obtained several lines of tobacco transformed with a trehalose-6-phosphate synthase gene of plant origin (Arabidopsis thaliana), involved in the first step of the biosynthesis of trehalose, a known osmoprotectant. Two showed distinct intensity of expression: high (B5H) and low (B1F). Such lines were analyzed for trehalose-6-phosphate content and the obtained results demonstrated to be in accordance with the expression results. In order to study the responses of photosynthesis to water deficit of transgenic lines in comparison to wild type (WT), three experiments were performed under different conditions: (1) Relative water (2) Leaf gas exchange (3) Modulated Chlorophyll a Fluorescence. Different responses in RWC of plant lines to water withdrawal were detected, with transgenic line B5H indicating less water loss after the water withdrawal period. Similar responses to water deficit regarding the leaf gas exchanges were recorded for the three lines. When subjected to water deficit stress situations, higher F _v/F _m, Φ_PSII and qP were detected for the transgenic lines. Under a SWC of 20% where higher values for such parameters were detected with special relevance for the B5H line, indicating a possible higher ability to withstand severe drought stress and to resist to prolonged periods without water than the B1F and WT lines.