Metabolomics - A robust bioanalytical approach for the discovery of the modes-of-action of pesticides: A review

The agrochemical industry is facing great undertaking that includes increasing demand for the development of new crop protection agents that are safe for the environment and the consumers, and at the same time combat the issue of the emergence of resistance pest strains. The mode-of-action (MoA) is among the features of a bioactive compound that largely determine whether the abovementioned issues are addressed or not, and subsequently whether its commercial development will be addressed. The early discovery of the MoA of bioactive compounds could accelerate pesticide research and development by reducing the required time and costs. Based on advances in synthetic and natural product chemistry, scientists have access to a vast number of compounds that could potentially be developed as crop protection agents. The screening of such compounds with respect to their MoA requires accurate and robust bioanalytical tools. Metabolomics is a powerful bioanalytical tool that will likely play a significant role in the acceleration of the discovery of MoA of bioactive compounds. In the present review, the capabilities and principles and applications of metabolomics for the study of the MoA of herbicides, insecticides, acaricides, fungicides, and antibiotics are discussed.     

大肠杆菌乙酰辅酶A羧化酶accD亚基表达载体的构建及遗传转化研究

为探索大肠杆菌异质型乙酰辅酶A羧化酶（ACCase）的β-CT亚基accD基因对油料作物含油量的影响,将目的基因E.coli accD（eaccD）与拟南芥Rubisco小亚基（rbcS）转运肽基因融合,构建以油菜Napin启动子驱动的种子特异表达载体。本试验应用农杆菌介导法,将该表达载体转入油菜,通过PCR检测获得了...     

Seasonal Patterns of Photosynthetic Traits in Early Maturing Maize

A biochemical approach to maize breeding must be based on an exact knowledge about changes of relevant traits with ontogenetic stage. In 1983 seasonal patterns of net assimilation rate (NAR), chlorophyll and carotenoids contents, and of activities of RuBP carboxylase, PEP carboxylase and NADP malate dehydrogenase were recorded for two early maturing maize cultivars. For all traits the general trends were similar for both cultivars throughout the growing season but short term fluctuations were less synchronized after anthesis. About 75% of the maximum green leaf area was expanded within 20 days around middle of July. Specific seasonal patterns were observed for all traits. Before anthesis values were maximum for NAR in late June and in early July, for enzyme activities at the end of June and for pigment contents at the beginning of July. First decreases occurred for all traits before anthesis, further decreases were observed for PEP carboxylase in August and for chlorophyll content in September. Marked temporary declines coincided for NAR and pigment contents with a cold spell end of June and for these traits and RuBP carboxylase activity with anthesis. Evidence was scarce that any of the photosynthetic traits reflected the photosynthetic efficience of the two cultivars for a long stretch of time.     

一些栽培作物的核酮糖-1,5-二磷酸羧化酶/加氧酶动力学特征

在相同条件下,对C_2植物中的9种双子叶植物和3种单子叶植物的核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)动力学特性进行了测定。K_m(CO_2)(K_c)和K_m(O_2)(K_(?))的变化范围分别为7.3～30μmol/L和300～1000μmol/L。变异系数分别为52％和38％。K_c值的变异明显高于K_o值。V_(max)(CO_2)(V_c)和V_(max)(O_2)(V_(?))分别为50～1500和16～500n mol/mg酶蛋白·min,变异系数相应为97％和87％。专一性因子的变化相对较小,为78～121,变异系数为15％。说明在C_3植物中,Rubisoo动力学特性的差异主要反应在V_(max)值上,核酮糖-1,5-二磷酸羧化酶(Rubisc)的动力学特性变化大于核酮糖-1,5-二磷酸加氧酶(Rubiso)。随着物种的进化,K_c、K_o值明显降低,而V_c、V_o值显著增加,致使Rubisco活性显著提高。Rubisco的羧化/加氧比也依物种的进化而上升。K_c与K_o、V_c与V_o之间具有显著的同步变化趋势,相关系数均超过极显著水平。     

甘蔗不同基因型2种光合酶的热稳定性

甘蔗不同基因型之间叶片 PEP 羧化酶的热稳定性有较大的差异。甘蔗叶片 PEP 羧化酶对高温敏感,在伸长盛期酶热稳定性相对较高;随着环境温度的下降,到伸长后期酶热稳定性明显变低,甘蔗叶片 NADP-苹果酸酶的热稳定性很高,不同基因型之间差异不大。显然,热逆境不会导致 NADP-苹果酸酶的严重失活而影响光合作用。     

四种油料作物中的细菌型PEPC基因的鉴定及在发育种子中的表达

鉴定和获取了四种油料作物（油菜、大豆、花生和芝麻）中的细菌型PEPC基因,分析了所编码蛋白的保守结构域（BOX I-IV）和蛋白作用功能位点。基因包括甘蓝型油菜的Bna10093361、Bna1009749和Bna10093360,大豆的Glyma10g34970.1, Glyma01g22840.1和Glyma02g14500.1,芝麻的SIN1018296和花生的AhPPC5。这8个基因含有19～21个内含子,内部插入一个约350～600bp的高度变异区,编码的蛋白在C端形成R/KNTG结构域,在N端缺乏磷酸化作用位点。在种子发育的不同时期,油菜中仅Bna10093360表达,但其表达量不到油菜BnActin表达量的0.1%;大豆中Glyma10g34970.1表达量最高（接近大豆GlymaActin的2%）,Glyma02g14500.1次之;花生AhPPC5表达量为花生AhActin的32%～175%,在种子不同发育时期表达量为早期>中期>晚期;芝麻SIN1018296表达量为芝麻SINActin的3%～18%,在种子发育时期的表达趋势和花生AhPPC5相似。8个基因种子中的表达模式差异明显,说明细菌型PEPC基因可能存在着广泛的功能分化。       

3种石斛PEPC羧化酶活力比较

 石斛属植物具有兼性景天酸代谢植物特征,磷酸烯醇式丙酮酸羧化酶是该类植物碳代谢的关键酶。本研究选用3种石斛为材料,测定了其磷酸烯醇式丙酮酸羧化酶活性。结果表明,报春石斛晚间黑暗条件下酶活性较强,凌晨高达25μmolCO2/(m2·s),而在中午光照最充足时,酶的活性相对较弱;金钗石斛酶活性日变化特点与报春石斛相似,但是活性比较弱;鼓槌石斛的酶活性很弱,通常在8μmolCO2/(m2·s)以下,昼夜变化幅度很小,表现为黑暗状态下比光下更强。     

重叠延伸PCR克隆拟南芥ACCase基因和植物表达载体构建

植物的乙酰辅酶A羧化酶(ACCase)催化乙酰辅酶A羧化生成丙二酸单酰辅酶A,是脂肪酸合成的第一步,该步骤是种子脂肪酸合成与含油量形成的关键调控步骤。拟南芥中的真核形式ACCase基因cDNA长约7,000bp,难以直接克隆。本研究先通过常规PCR将其cDNA分成八个重叠的片段分别扩增出来,再用重叠延伸PCR将得到的片段逐步拼接成长度为6890bp的完整的基因序列,测序证实克隆序列正确无误,最后将其克隆到农杆菌转化植物的载体上。序列分析发现ACCase基因的开放阅读框长度为6765bp,编码一个含2254个氨基酸残基的多肽链,推测其分子量约为250KDa。ACCase基因的成功克隆为利用该基因调控油料作物的含油量奠定了良好基础,同时为克隆长度较大的基因提供了一种经济可行的方法。     

水稻叶片自然衰老过程中Rubisco大亚基的含量变化

 以水稻低叶绿素b突变体及其野生型（镇恢249）为材料,研究了水稻第5叶叶片自然衰老过程中光合放氧、Rubisco大亚基含量变化以及对胰蛋白酶敏感性的变化,并通过喷施不同的抗氧化剂以及氧化剂研究其对Rubisco大亚基含量变化的影响。结果表明,叶片全展后随着衰老进程,突变体与野生型光合放氧速率、Rubisco大亚基相对含量呈下降趋势,抗氧化剂可以不同程度地缓解Rubisco大亚基含量的下降,而氧化剂则加速Rubisco大亚基含量的下降。低叶绿素b突变体的光合放氧速率高于野生型,Rubisco大亚基含量下降慢于野生型,并且衰老进程中突变体Rubisco大亚基对胰蛋白酶的敏感性低于野生型,推测活性氧是Rubisco降解的调节剂之一。     

The response of ACCase-resistant Phalaris paradoxa populations involves two different target site mutations

Phalaris paradoxa (awned canary-grass) is an aggressive annual winter weed in wheat and other arable crops that is controlled mainly by ACCase-inhibiting herbicides: cyclohexanediones (DIMs), aryloxyphenoxypropionates (FOPs) and phenylpyrazolines (DENs, e.g. pinoxaden). The selection pressure imposed on the weed populations by repeated use of these herbicides has resulted in the evolution of increased numbers of ACCase-resistant populations of P. paradoxa in Israel and other countries. Two populations, Revadim (RV) and Mishmar Ha'emek (MH) that were exposed to differing weed and crop management tactics were investigated. Both populations were highly resistant to all FOPs, pinoxaden and cycloxydim, but responded differently to some DIMs. RV plants exhibited much higher resistance to tralkoxydim than MH plants, while showing similar low levels of resistance to tepraloxydim and clethodim. Both populations were equally susceptible to graminicides with other modes of action. The mutations responsible for the observed resistance were identified using PCR-RFLP and by sequencing the carboxyl transferase domain of the chloroplastic ACCase gene. RV plants possess a substitution of Asp₂₀₇₈ to Gly, whereas in MH population a mixture of Ile₂₀₄₁ to Asn or Asp₂₀₇₈ to Gly was found. Our study demonstrates that lack of herbicide and crop rotation may result in the evolution of diverse target site mutations and differential response of the whole plant to ACCase inhibitors.