棉花根系和叶片质外体汁液分离方法的改进

质外体汁液(apoplastic washing fluid, AWF)在植物生长发育和抵抗生物及非生物逆境方面发挥着重要作用。目前普遍采用真空渗透离心法提取质外体汁液,但具体提取流程和条件却因植物培养条件、种类和器官等不同而不同。本试验以营养液培养的棉花幼苗为材料,改进了棉花根系和叶片AWF分离的取样方法、渗透条件和离心参数等。结果表明,相对于通常的非整体取样,改良后整体取样简单易操作且显著降低了质外体与共质体的苹果酸脱氢酶(malate dehydrogenase, MDH)活性比值,更适宜开展质外体汁液组分研究。根据真空渗透鲜重增加和AWF稀释因子,进一步证明根系不用真空渗透;而叶片需真空渗透,适宜真空强度/时间为-60 kPa/1min,真空后恢复到正常压强约110s。证明叶片颜色变深面积可作为判定真空渗透强度或时间是否适宜的一种简易可行方法。最后,AWF体积、质外体与共质体的可溶性蛋白含量比值和MDH活性比值综合分析表明,棉花根系适宜离心力大小/时间长度为800×g/10～20 min,叶片为400×g/5 min。本改进方法为棉花AWF组分,如蛋白质组学和代谢组学等研究结果的准确性和可靠性奠定了基础,为其他作物AWF分离方法的优化提供了参考。     

改性尿素硝酸铵溶液调控氮素挥发和淋溶的研究

为了提高肥料的利用率,以尿素硝酸铵溶液为原料、聚氨酸为保护剂,复合抑制剂NBPT(N-丁基硫代磷酰三胺)和DMPP(3,4-二甲基吡唑磷酸盐)为材料,开发出改性尿素硝酸铵溶液(YUL1和YUL2),研究其对华北平原夏玉米追肥过程中的氨挥发和淋溶损失的调控效果。田间试验设置6个处理:不施氮肥(CK)、农民习惯追施尿素(CN)、优化追施尿素(CNU)、优化追施尿素硝酸铵溶液(UAN)、优化追施改性尿素硝酸铵溶液(YUL1)和优化追施改性尿素硝酸铵溶液(YUL2)。采用扫描电镜和能谱仪分析相关指标变化,在夏玉米喇叭口期追施氮肥后15d内进行田间原位连续动态观测氨挥发和土壤铵态氮和硝态氮变化,并在玉米成熟期测定产量,计算经济效益。结果表明,改性尿素硝酸铵溶液清澈无杂质,流延后成膜表面光滑、致密,抑制剂在膜表面分布均匀;能谱测试膜层表面磷硫含量增高,证明复合抑制剂与尿素硝酸铵溶液达到有效融合。在同等优化施氮量下:与CNU相比, YUL1氨挥发总量显著降低19.3%, YUL2增加9.6%;与UAN相比, YUL1、YUL2分别显著降低57.3%和42.0%。与其他施氮处理相比, YUL1和YUL2夏玉米季生长中后期0～20 cm土层依然保持相对较高的氮素含量水平,夏玉米收获后土壤硝态氮含量分别比CNU高46.0%和43.4%,比UAN高45.6%和44.7%;180～200cm土层硝态氮含量显著低于其他处理。在保证产量和净收益的同时,改性尿素硝酸铵肥料显著降低了氮素的氨挥发和淋溶损失浓度,尿酶抑制剂含量相对较高的YUL1抑制氨挥发的效果更好,硝化抑制剂含量相对高的YUL2硝态氮向下淋失的风险更小。     

谷子MDH基因非生物逆境响应特性研究

为揭示谷子MDH基因对逆境胁迫的响应,本研究通过序列比对得到2个谷子苹果酸脱氢酶(malate dehydrogenase, MDH)基因SiMDH1、SiMDH2,并对SiMDH1和SiMDH2的编码蛋白特征、基因结构、功能、进化等性状进行了系统的分析和预测,用荧光实时定量PCR(qPCR)检测了SiMDH1和SiMDH2在苗期不同逆境及关键生育期干旱胁迫和不同光照强度下的表达。蛋白特征参数和序列分析表明,SiMDH1和SiMDH2蛋白特征参数相似度很高,序列非常保守,都含有MDH基因的特征功能域苹果酸酶NAD结合域和苹果酸酶C端功能域。亚细胞定位预测显示,SiMDH1和SiMDH2主要定位于叶绿体、细胞质和线粒体。启动子区域顺式元件分析发现,在SiMDH1和SiMDH2启动子区域含有大量光应答、激素类和其他类应答元件。苗期逆境qPCR表达谱分析发现,SiMDH1和SiMDH2受脱落酸(ABA)、聚乙二醇(PEG)、高盐和低温不同程度诱导,揭示SiMDH1和SiMDH2参与了谷子苗期非生物逆境胁迫应答。进一步分析发现,SiMDH1和SiMDH2参与了谷子在拔节、抽穗、灌浆期的干旱应答,而光照强度会显著影响SiMDH1和SiMDH2基因在不同生育期的表达。本研究为进一步分析MDH逆境应答机制以及利用基因工程方法改善作物抗逆性提供了试验和理论支持。     

黄河三角洲冲积平原湿地土壤氧化还原酶活性与养分通径分析

运用通径分析法研究黄河三角洲冲击平原湿地土壤中氧化还原酶活性与土壤养分的关系。结果表明:土壤氧化还原酶活性关于土壤化学性质的标准化多元线性回归方程分别可以解释81.65%的过氧化氢酶活性变化和86.2%的过氧化物酶活性变化,剩余通径系数为0.428 4和0.371 5。土壤有机质成为影响冲积平原湿地土壤氧化还原酶活性最主要的因素。     

猪氨基酸代谢节俭机制新假说

猪尿氮排放量为总氮排放量的60%~70%,而尿素是尿液中的主要含氮物,其合成速率在很大程度上决定着尿氮以及总氮的排放量。因此,降低猪肝脏尿素合成速率是减少氮排放量的根本途径。本文首先介绍了当前猪氮减排常用的营养调控技术,然后分别就肝脏尿素合成的直接前体物(氨)与间接前体物(如甘氨酸和丙氨酸)以及氨基酸代谢燃料功能替代机制进行论述,在此基础上提出猪氨基酸代谢节俭机制新假说,即促进丙酮酸/葡萄糖等物质的供能效率,以降低谷氨酸等氨基酸的代谢速率,从而达到减少门静脉尿素前体物净流量、肝脏尿素合成以及尿氮排放量的目的。     

昆虫取食和剪叶刺激对油松针叶内部分防御物质的诱导效应

为了研究自然条件下昆虫取食及剪叶刺激对油松诱导抗性的影响,本试验应用香草醛—盐酸法,亚硝酸钠—硝酸铝比色法以及紫外分光光度法,分析了剪叶和不同程度油松毛虫取食处理后,混交林和纯林中油松针叶内部分次生代谢物和蛋白酶抑制剂活性的动态变化。结果表明:剪叶及不同程度油松毛虫取食能够诱导缩合单宁、黄酮含量和胰蛋白酶抑制剂、胰凝乳蛋白酶抑制剂活性增加。与对照相比,胰凝乳蛋白酶抑制剂在混交林中的活性比纯林更为明显。说明剪叶及昆虫取食可诱导增加油松针叶内的缩合单宁和黄酮含量、提高胰蛋白酶抑制剂和胰凝乳蛋白酶抑制剂的活性,进而增强油松的抗虫性,林分类型可在一定程度上影响油松诱导防御物质的变化。     

Screening and Analysis of Proteins Interacting with TaPDK from Physiological Male Sterility Induced by CHA in Wheat

To further research the regulatory network of pyruvate dehydrogenase kinase (designated as TaPDK) in physiological male-sterility (PHYMS) of wheat induced by chemical hybridizing agent (CHA) SQ-1, an anther cDNA library was constructed, and the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR. Yeast-two hybrid screening was performed by mating yeast strain Y2HGold containing BD-TaPDK bait plasmid with yeast strain Y187 including anther cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Ade/-His/-Leu/-Trp) (QDO), and further were incubated on QDO medium containing AbA and X-α-Gal. The interactions between TaPDK and the proteins obtained from positive colonies were further confirmed by co-transformation validation. After plasmids DNA were extracted from blue colonies and sequenced, the sequences results were analyzed by bioinformatic methods. Finally, 24 colonies were obtained, including eight genes, namely non-specific lipid-transfer protein precursor (TanLTP), polyubiquitin (TaPUbi), glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen (TaPCNA), CBS domain containing protein (TaCBS), actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and three new genes with unknown function. The results of quantitative RT-PCR showed that the expression levels of TanLTP, TaPUbi, and TaPCNA were obviously up-regulated in PHYMS anther, and TaCBS expression was only increased at the tricellular stage in PHYMS anther compared with in fertile lines. Whereas, the expression of TaPDK was obviously down-regulated in PHYMS lines. Collectively, these datas indicated that the majority of candidate proteins might be related to pollen abortion in PHYMS lines, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating pyruvate dehydrogenase complex activity.     

Influence of soil factors on the soil enzyme inhibition by Cd

A comparative study was conducted on the toxicity of Cd to alkaline phosphatase activity (ALP) and dehydrogenase activity (DHA) in 18 top soils with contrasting soil properties representative of 14 major soil types in China. Soil pH and carbonate content, soil organic matter, and cation exchange capacity (CEC) largely affected the Cd toxicity on two enzyme activities; with the soil pH having only minor effect on the median ecological dose values based on total Cd concentrations (ED_{50 T}). The values of ED_{50 T}/ED_{50 W} (based on water-soluble Cd content) of alkaline phosphatase and dehydrogenase were strongly influenced by pH and CEC contents, which explained up to 71% of the variation for alkaline phosphatase, 82% of the variation for dehydrogenase, and also were significantly correlated with the parameter K_F derived from Freundlich adsorption isotherms. This study suggests that the values of ED_{50 T}/ED_{50 W} could be useful to evaluate the buffer capacity of soils which protects soil enzymes from harmful effects of heavy metal.     

微透析法获取鹅掌柴嫩茎质外体汁液的研究

该文研究的重点是获取质外体汁液。假设微透析技术可以在植物上得到质外体汁液,并以盆栽鹅掌柴为材料,在活体嫩茎插入微透析探针后的不同时间段内分别收集透析液,测定其中的离子浓度和苹果酸脱氢酶活性加以验证。结果表明:在探针插入120 min后,收集的透析液中Na+、K+和Ca2+浓度逐渐趋于平缓,并且苹果酸脱氢酶的活性消失,从而证明120 min后得到的微透析液,没有受到破损细胞液的污染,是纯净的鹅掌柴嫩茎质外体汁液，从而为活体、方便、快捷地获取植物质外体汁液提供新的方法。      

Aggressiveness of Xanthomonas axonopodis pv. glycines isolates to soybean and hypersensitivity responses by other plants

Xanthomonas axonopodis pv. glycines ( Xag ) causes bacterial pustule disease which can significantly reduce the production of soybean. A collection of 26 isolates of Xag from different soybean-production areas of Thailand was shown to differ with regard to aggressiveness on soybean. They also differed in their ability to induce a hypersensitive response (HR) on four cultivars of tobacco and on other plant species including pepper, tomato, cucumber, pea and sesame. Tomato was most sensitive to HR induction by Xag . Isolate KU-P-34017 caused an HR on all the plant species tested. The minimal concentration of KU-P-34017 needed to induce HR on tobacco was approximately 5 × 10⁸ CFU mL⁻¹. A bacterium–plant interaction period of at least 2·5 h was necessary for HR, and different temperatures, relative humidity and light periods did not affect HR development. Inhibitors of eukaryotic metabolism, including cobalt chloride, lanthanum chloride and sodium orthovanadate (completely), and cycloheximide (partially) blocked the HR on tobacco, indicating the association of an active plant response. In contrast, the HR on tomato was inhibited only by cobalt chloride.