过表达ZmIBH1-1提高玉米苗期抗旱性

【目的】干旱是严重影响玉米生长发育进程的一个重要因素。挖掘玉米抗旱相关基因,通过转基因功能验证和转录组分析,解析关键基因在响应干旱胁迫过程中的分子调控机制,为抗旱分子育种和遗传改良提供理论依据。【方法】以玉米自交系B104（WT）为背景材料,利用农杆菌介导方法构建过表达ZmIBH1-1转基因株系（ZmIBH1-1-OE）;通过对转基因植株进行草铵膦抗性筛选、标记基因和目的基因PCR检测,以及运用实时荧光定量PCR检测目的基因的表达情况,鉴定阳性植株和株系;以WT和ZmIBH1-1-OE转基因株系为材料,通过干旱处理（20% PEG6000）,进行表型鉴定和耐旱生理生化指标测定,验证ZmIBH1-1的抗旱功能;通过对干旱胁迫下玉米4叶期转录组的比较分析,鉴定出差异表达的基因（differentially expressed genes,DEGs）;结合DAP-seq（DNA affinity purification sequencing）分析,初步确定ZmIBH1-1蛋白直接调控与抗旱相关的下游靶基因,利用基因组可视化软件IGV（integrative genomics viewer）分析ZmIBH1-1蛋白结合候选靶基因的位置,然后通过Dual-Luciferase试验验证ZmIBH1-1蛋白与靶基因的调控关系。【结果】通过玉米遗传转化获得12个转化事件;T₃代中,能同时检测到标记基因Bar和目的基因ZmIBH1-1的植株有458个,实时荧光定量PCR检测结果表明,ZmIBH1-1-OE中ZmIBH1-1的表达量显著高于WT,株系3和株系8表达量最高,将其自交获得T₄代转基因株系用于后续试验。在干旱胁迫条件下,ZmIBH1-1-OE株系存活率、叶片相对含水量、叶绿素含量、可溶性蛋白含量及其生理生化指标（超氧化物歧化酶、过氧化物酶、过氧化氢酶活性）均显著高于WT,说明玉米中过量表达ZmIBH1-1赋予玉米更高的耐旱性。转录组分析结果表明,WT与ZmIBH1-1-OE株系在干旱胁迫下有1 214个差异表达基因;Gene Ontology（GO）功能富集分析结果表明,差异表达基因主要涉及生物过程、细胞组分和分子功能,如在生物过程中主要涉及到光合作用、应激响应、脱水响应等;KEGG富集分析表明,差异表达基因主要参与植物激素信号传导、新陈代谢等过程。结合转录组显著差异表达基因和DAP-Seq分析所得到ZmIBH1-1蛋白的靶基因,初步确定ZmIBH1-1蛋白直接调控与抗旱相关的11个候选靶基因,包括2个钙信号相关基因、3个半胱氨酸代谢相关基因、1个bHLH转录因子、1个应激响应蛋白、1个谷胱甘肽转移酶、1个氧化还原过程蛋白和2个乙烯响应因子;基因组可视化结果显示ZmIBH1-1蛋白可以结合靶基因启动子区;随后通过Dual-Luciferase试验进一步表明,ZmIBH1-1蛋白可以直接作用于11个候选靶基因,其中,ZmIBH1-1蛋白可以促进ZmCa-M、ZmSYCO、ZmbHLH54、ZmGlu-r1、ZmCLPB3和ZmP450-99A2的表达,抑制ZmAGD12、ZmCYS、ZmCYSB、ZmERF-107和ZmEIN3的表达。此外,在干旱胁迫下NAC、WRKY、MYB等转录因子在ZmIBH1-1-OE和WT株系中也存在差异表达。【结论】ZmIBH1-1的过表达可以增强玉米苗期的耐旱性;ZmIBH1-1蛋白通过直接调控乙烯信号通路中的ZmERF-107和ZmEIN3的表达提高玉米的耐旱性;ZmIBH1-1蛋白通过直接调控钙信号相关基因ZmCa-M和ZmAGD12增强玉米的耐旱性;ZmIBH1-1蛋白可能通过间接调控NAC、WRKY、MYB等转录因子响应干旱胁迫。     

Exogenous application of allelopathic water extracts helps improving tolerance against terminal heat and drought stresses in bread wheat (Triticum aestivum L. Em. Thell.)

The allelopathic water extracts (AWEs) may help improve the tolerance of crop plants against abiotic stresses owing to the presence of the secondary metabolites (i.e., allelochemicals). We conducted four independent experiments to evaluate the influence of exogenous application of AWEs (applied through seed priming or foliage spray) in improving the terminal heat and drought tolerance in bread wheat. In all the experiments, two wheat cultivars, viz. Mairaj‐2008 (drought and heat tolerant) and Faisalabad‐2008 (drought and heat sensitive), were raised in pots. Both wheat cultivars were raised under ambient conditions in the wire house till leaf boot stage (booting) by maintaining the pots at 75% water‐holding capacity (WHC). Then, managed drought and heat stresses were imposed by maintaining the pots at 35% WHC, or shifting the pots inside the glass canopies (at 75% WHC), at booting, anthesis and the grain filling stages. Drought stress reduced the grain yield of wheat by 39%–49%. Foliar application of AWEs improved the grain yield of wheat by 26%–31%, while seed priming with AWEs improved the grain yield by 18%–26%, respectively, than drought stress. Terminal heat stress reduced the grain yield of wheat by 38%. Seed priming with AWEs improved the grain yield by 21%–27%; while foliar application of AWEs improved the grain yield by 25%–29% than the heat stress treatment. In conclusion, the exogenous application of AWEs improved the stay green, accumulation of proline, soluble phenolics and glycine betaine, which helped to stabilize the biological membranes and improved the tolerance against terminal drought and heat stresses.     

Determination of critical nitrogen dilution curve based on leaf area index for winter wheat in the Guanzhong Plain,Northwest China

Excessive use of nitrogen(N) fertilizers in agricultural systems increases the cost of production and risk of environmental pollution. Therefore, determination of optimum N requirements for plant growth is necessary. Previous studies mostly established critical N dilution curves based on aboveground dry matter(DM) or leaf dry matter(LDM) and stem dry matter(SDM), to diagnose the N nutrition status of the whole plant. As these methods are time consuming, we investigated the more rapidly determined leaf area index(LAI) method to establish the critical nitrogen(N_c) dilution curve, and the curve was used to diagnose plant N status for winter wheat in Guanzhong Plain in Northwest China. Field experiments were conducted using four N fertilization levels(0, 105, 210 and 315 kg ha-1) applied to six wheat cultivars in the 2013–2014 and 2014–2015 growing seasons. LAI, DM, plant N concentration(PNC) and grain yield were determined. Data points from four cultivars were used for establishing the N_c curve and data points from the remaining two cultivars were used for validating the curve. The N_c dilution curve was validated for N-limiting and non-N-limiting growth conditions and there was good agreement between estimated and observed values. The N nutrition index(NNI) ranged from 0.41 to 1.25 and the accumulated plant N deficit(N_(and)) ranged from 60.38 to –17.92 kg ha~(-1) during the growing season. The relative grain yield was significantly affected by NNI and was adequately described with a parabolic function. The N_c curve based on LAI can be adopted as an alternative and more rapid approach to diagnose plant N status to support N fertilization decisions during the vegetative growth of winter wheat in Guanzhong Plain in Northwest China.     

欧洲葡萄‘粉红亚都蜜’NAC基因DRL1负向调节植物抗旱性

以欧洲葡萄‘粉红亚都蜜’（Vitis vinifera‘Yatomo Rose’）为材料，利用荧光定量PCR技术和转基因技术研究葡萄NAC转录因子DRL1基因对逆境的响应。欧洲葡萄‘粉红亚都蜜’在激素和逆境胁迫下，DRL1表达呈下降趋势，其中以ABA和干旱胁迫处理最为显著。在ABA处理下DRL1转基因烟草株系种子萌发率和根长均高于野生型。干旱处理下，转基因植株对干旱的耐受性降低，同时胁迫相关基因NtLEA5、NtP5CR1、NtPSCS1、NtERD10C和NtDREB3的表达水平比野生型显著下降。此外，DRL1转基因烟草茎中柱发育受到抑制，尤其是导管横切面积仅为野生型的58%。以上结果表明，DRL1基因可能作为1个负向调节子参与植物的干旱胁迫。     

Impact of soil water content on maize responses to the plant growth-promoting rhizobacterium Azospirillum lipoferum CRT1

Members of the bacterial genus Azospirillum are root-associated bacteria that increase yield in cereals by promoting growth and alleviating drought stress. How plants integrate the many bacterium-derived growth-promoting stimuli with other environmental factors to generate a coordinated response remains unresolved. Using a commercial Azospirillum strain, A. lipoferum CRT1 and two host maize cultivars, it was observed that bacterization reduced the drought-induced increase in lateral root growth and enhanced the flood-induced increase in lateral root growth in the more drought- and flood-sensitive cultivar. In the other one, A. lipoferum CRT1 only elicited a moderate root growth response under low soil water potential. The photosynthetic potential and activity were increased in the earlier cultivar and decreased in the later one, irrespective of the soil water content. No impact of the bacterium was seen on the growth of the leaves of both cultivars under both stresses until the third leaf stage, therefore suggesting that it is a consequence of multiple primary adaptations to biotic and abiotic stresses. It is suggested that host–bacteria recognition leads to a stress-specific modulation of the root response and a differential stress-independent effect on photosynthesis. This is the first report of the impact of Azospirillum under flood conditions.     

Stoffbildung, CO2-Gaswechsel, Kohlenhydrat- und Stickstoffgehalt von Winterweizen bei erhöhter CO2-Konzentration und Trocken-streß

Dry Matter Production, CO₂ Exchange, Carbohydrate and Nitrogen Content of Winter Wheat at Elevated CO₂ Concentration and Drought Stress
Methods of mathematical modelling and simulation are being used to an increasing degree in estimating the effects of rising atmospheric CO₂ concentration and changing climatic conditions on agricultural ecosystems. In this context, detailed knowledge is required about the possible effects on crop growth and physiological processes. To this aim, the influence of an elevated CO₂ concentration and of drought stress on dry matter production, CO₂ exchange, and on carbohydrate and nitrogen content was studied in two winter wheat varieties from shooting to milk ripeness. Elevated CO₂ concentration leads to a compensation of drought stress and at optimal water supply to an increase of vegetative dry matter and of yield to the fourfold value. This effects were caused by enhanced growth of secondary tillers which were reduced in plants cultivated at atmospheric CO₂ concentration. Analogous effects in the development of ear organs were influenced additionally by competitive interactions between the developing organs. The content and the mass of ethanol soluble carbohydrates in leaves and stems were increased after the CO₂ treatment and exhausted more completely during the grain filling period after drought stress. Plants cultivated from shooting to milk ripeness at elevated CO₂ concentration showed a reduced response of net photosynthesis rate to increasing CO₂ concentration by comparison with untreated plants. The rate of dark respiration was increased in this plants.     

冻融中土壤的水热动态

根据在北京郊区的试验,认为土壤水冻融过程可分为三阶段,其中第二阶段有聚墒现象,平均聚墒8.6mm,所聚的墒在融化过程中又逐渐消失。气温回升至-2℃左右是冻层下部由冻到融的转折温度。冬灌水保存到来年春季的数量有限,本试验中1m 土层内平均不足20mm。     

Effect of Completion of Vernalization on Generative Development of Winter Rape (Brassica napus L. var. oleifera) Plants

Germinating seeds and young plants of winter rape var. Górczañski were vernalized for 56–63 days under conditions of 9-hour day, at the temperature 2 and 5 °C and in continuous darkness at the temperature 2 °C. After vernalization the plants grew under conditions enabling to complete vernalization: in a glass-house at the temperature day/night 15/10 °C and in semi natural conditions of open vegetation hall in the period from June till August. After sub-optimal vernalization further growth of the plants at lowered temperature increased its effectiveness (completion of vernalization). Depending on the degree of the vernalization of the plants the completion of their vernalization was both obligatory, i.e. conditioning the acquisition of the ability of generative development, and facultative i.e. accelerating this development. It has been demonstrated that the population of plants of the examined variety is strongly differentiated not only with respect of vernalization requirements in the particular plants, but also what regards the effectiveness of vernalization completion. New observations have been made indicating that the mechanisms controlling the successive phases of generative development, i.e. phase of forming flower buds and the flowering phase are not identical which may be interpreted as indicating that the "flowering factor" is polymorphous.