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.     

内生菌与雷公藤细胞悬浮共培养的生理生化特性

将2株雷公藤内生真菌Fusarium oxysporum NS33与Penicillium steckii NS6、2株内生细菌Enterobacter cloacae sub sp LG3与Serratia marcescens LY1及其组合分别与雷公藤细胞悬浮共培养,对不同培养体系内雷公藤细胞的生长及其生理生化特征进行研究。结果显示,在共培养前期,与对照相比,接种单一内生菌株提高了细胞的干重,其中菌株NS6的促生效果最明显;而在共培养后期,无论是单一内生菌还是混合内生菌均对细胞生长具有抑制作用。内生真菌和菌株组合处理的培养液p H值有明显的升高,而内生细菌LY1则明显降低了培养液的p H值,其具有产酸性。另外,当雷公藤细胞同混合菌株共培养时,培养基中总糖消耗量是最大的,而接种单独菌株时则对细胞可溶性蛋白含量具有一定的提高作用。接种内生菌会影响雷公藤细胞的POD、CAT及SOD活性,与对照相比,接种单独菌株会更加提升POD与CAT的活性,而细胞MDA含量则明显下降。     

1-MCP处理对“丽格”海棠果实采后生理及品质的影响

以"丽格"海棠果实为试材,研究经1.0μL/L的1-MCP处理后,室温(20±1)℃条件下贮藏的海棠果实品质和生理变化,并利用主成分对采后测定的各项生理指标进行分析。结果表明,与不加1-MCP的对照组相比,1-MCP处理可保持海棠果实硬度,抑制可溶性固形物(TSS)含量的升高,减缓VC含量的降低、果皮叶绿素的分解和过氧化物酶(POD)活性的下降,降低呼吸强度和乙烯生成速率,有效地延缓果实衰老,使海棠果更耐贮藏。主成分分析在特征值大于1时将经1-MCP处理后的海棠果实8个生理指标综合为2个因子,累积方差贡献率达到84.44%,其中第1主成分主要代表果肉硬度和叶绿素信息,第2主成分主要代表乙烯释放量信息。     

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。     

异源表达玉米ZmC3H54基因增强水稻的耐旱性

CCCH锌指蛋白是一类重要的转录调控因子。从玉米中分离得到一个受干旱和ABA诱导表达的CCCH型锌指蛋白基因ZmC3H54，通过构建过量表达载体并转化水稻来进一步研究其功能。 与对照组相比，转基因植株在干旱胁迫处理下具有更高的相对含水量与存活率以及较低的相对电导率，表明过量表达ZmC3H54基因可以提高转基因水稻的耐旱性。此外，转基因水稻幼苗对外源ABA敏感性更高。以上结果表明玉米ZmC3H54基因可能是通过ABA信号通路调控植物对干旱的耐受性。     

不同施氮水平对柳枝稷光合特性及抗旱性的影响

为了探究宁夏银北盐碱地区柳枝稷高产优质高效栽培过程中最佳的施氮量及其对柳枝稷叶片光合特性及抗旱性的影响,本研究采用大田试验,以Cave-in-Rock品种柳枝稷为供试材料,设无氮添加(0 kg·hm-2,N0)、施低氮(60 kg·hm-2,N60)、中氮(120 kg·hm-2,N120)和高氮(240 kg·hm-2,N240)共4个施氮水平,分析比较了各生育时期内柳枝稷叶片光合特性、渗透调节物质及抗氧化酶活性的变化,同时采用隶属函数法综合评价了盐碱地柳枝稷的抗旱性.结果表明,随着不同施氮水平的增加,柳枝稷各生育时期内叶片叶绿素相对含量(SPAD)、净光合速率(Pn)、气孔导度(Gs)和细胞间隙CO2浓度(Ci)整体呈现先升后降的趋势,均在施中氮(N120)处理下达到峰值.与无氮添加(N0)处理相比,施低氮(N60)、中氮(N120)和高氮(N240)处理下柳枝稷叶片的SPAD值平均增加了4.73％、18.71％和8.86％,净光合速率(Pn)平均提高了5.55％、17.02％和12.41％,气孔导度(Gs)平均升高了7.87％、56.18％和39.33％,细胞间隙CO2浓度(Ci)平均增加了7.86％、30.71％和13.81％.柳枝稷叶片蒸腾速率(Tr)在高氮(N240)处理下达到最大值,叶片水分利用效率(WUE)随着不同施氮水平的增加而逐渐下降.隶属函数分析结果表明,施中氮(N120)处理下柳枝稷各抗旱指标隶属函数值的均值最大.因此,本试验条件下有利于提高柳枝稷叶片光合能力和抗旱性的适宜施氮水平为120 kg·hm-2.     

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.     

Influencing in vitro clonal propagation of Chonemorpha fragrans(moon) Alston by culture media strength,plant growth regulators,carbon source and photo periodic incubation

Chonemorpha fragrans is an endangered medicinal woody climber,regarded among alternative plant sources of camptothecin.Camptothecin is a monoterpene indole anti-cancer alkaloid with annual trade value of over three billion U.S.dollars in the recent,and is used in the production of its analog drugs approved for the chemotherapy of cancer of varied types.Effects of plant growth regulators,culture media strength and photoperi-odic duration on the micropropagation ef ciency of C.fragrans from nodal segment explants were studied on Murashige and Skoog(MS)medium amended with Thidiazuron(TDZ),Benzylaminopurine(BAP)or Kinetin(Kin).Thidiazuron was more ef cient over BAP and Kin when half basal MS medium was used over full or quarter strength.Results of carbon source experiment showed sucrose as the most effective over glucose,fructose,and maltose in the clonal production.Studies on the photope-riodic incubation duration showed 12 h as the best light period and sub or supra-optimal resulted in the production of abnormal and albino micro shoots.Experimental results on the evaluation of physiological,biochemical parameters showed the role of pigment molecules and antioxidant systems in the production of albino micro shoots.     

干旱胁迫下不同品种杨树矿质离子含量变化

【目的】了解杨树在干旱胁迫下矿质离子分布及其动态变化。【方法】以吴屯杨、新疆杨和荷兰杨为实验材料,在中度和重度干旱条件下测定了根、茎、叶和质外体的K^+、Ca^2+、Na^+和Mg^2+等4种离子含量的变化。【结果】干旱胁迫使K^+、Ca^2+和Na^+离子含量上升,Mg^2+含量下降,其中K^+和Ca^2+含量自胁迫开始明显升高,Na^+含量则在中度胁迫之后明显增加。离子在各器官分布情况为:K^+主要分布于杨树的叶片,Ca^2+、Na^+和Mg^2+主要分布于杨树根部,且离子分布存在品种间差异,荷兰杨和新疆杨中K^+分布为:叶>根>茎,而吴屯杨体内K^+分布则为:叶>茎>根;3种杨树中Ca^2+分布均为:根>叶>茎;吴屯杨和荷兰杨中Na^+分布为:根>茎>叶,新疆杨则为:根>叶>茎;在Mg^2+的分布上吴屯杨和新疆杨较为类似即:根>叶>茎,而荷兰杨为叶>根>茎。【结论】干旱胁迫下杨树首选吸收的离子是K^+和Ca^2+,K^+主要分布在杨树的叶片中,其次是根部,Ca^2+主要分布在杨树的根部。重度干旱胁迫下,杨树开始吸收Na^+,储存在杨树的根部。不同品种杨树之间离子的分布和吸收都存在品种差异。质外体作为离子的运输通道,在各干旱条件下运输4种矿质离子至杨树的其他部位,Mg^2+存在滞留在质外体中的情况。本研究可为植物在干旱胁迫下矿质离子吸收和分布的研究提供理论依据。