Bioregulatory Effects of the Fungicidal Strobilurin Kresoxim-methyl in Wheat (Triticum aestivum)

Apart from its fungicidal effect, the strobilurin kresoxim-methyl (BAS 490 F) was found to induce physiological and developmental alterations in wheat (Triticum aestivum L.) which are seen in connection with improved yield. In a series of biotests including heterotrophic maize and photoautotrophic algal cell suspensions, duckweed, isolated mustard shoots and germinating cress seeds, kresoxim-methyl showed a similar response pattern to standard auxins (e.g. indol-3-ylacetic acid, IAA; 2-(1-naphthyl)acetic acid, α-NAA). Auxin-like activity of kresoxim-methyl was also found when stem explants of tobacco were cultured on a hormone-free medium. Kresoxim-methyl stimulated shoot formation, particularly at 10^-7 M . The same effect was induced by 10^-8 M IAA. The determination of phytohormone-like substances in shoots of wheat plants foliar-treated with 7×10^-4 M kresoxim-methyl revealed only slightly changed levels of endogenous IAA, gibberellins and abscisic acid. In contrast, the contents of dihydrozeatin riboside-type cytokinins increased to 160% of the control, while trans-zeatin riboside- and isopentenyladenosine-type cytokinins remained nearly unchanged. The most remarkable alterations were the reductions in 1-aminocyclopropane-1-carboxylic acid (ACC) levels and ethylene formation which were demonstrated in intact plants, leaf discs and the shoots of wheat subjected to drought stress. Kresoxim-methyl affected the induction of ACC synthase activity which converts S-adenosyl-methionine to ACC in ethylene biosynthesis. In shoots from foliar-treated wheat plants, 10^-4 M kresoxim-methyl inhibited stress-induced increases in endogenous ACC synthase activity, ACC levels and ethylene formation by approximately 50%. Reductions in ACC synthase activity and ACC levels of 30% were also obtained at low concentrations of α-NAA (10^-6 M ). In contrast, ACC synthase activity in vitro was not influenced by adding the compounds. In wheat leaf discs, the inhibiting effect of kresoxim-methyl, α-NAA and IAA on ethylene formation was accompanied by delayed leaf senescence, characterized by reduced chlorophyll loss. However, in contrast to kresoxim-methyl which showed only inhibitory activity on ethylene synthesis over a wide range of concentrations applied, the auxins stimulated ethylene production at high concentrations of about 10^-4 M . The inhibition of ethylene biosynthesis by kresoxim-methyl, together with an increase in endogenous cytokinins could explain the retardation of senescence and the intensified green leaf pigmentation in wheat exposed to this strobilurin. © 1997 SCI.     

桃ACC氧化酶cDNA片段扩增及其克隆和鉴定

采用“富集ＰＣＲ”方法,用单个特异引物和ｏｌｉｇｏ（ｄＴ）１５从桃伤处理叶片ｃＤＮＡ 中扩增出特异性片段⒚将扩增产物分离并克隆筛选到１３ 个重组克隆,其中有６ 个克隆能与ＡＣＣ氧化酶基因组ＤＮＡ 杂交,对其中一个阳性克隆ｐＧＥＭＡＣ１２ 作酶切分析,发现其酶切图谱与已报道的桃果实成熟相关的ＡＣＣ氧化酶ｃＤＮＡ 基本一致⒚ＤＮＡ 序列分析表明,插入片段两端ＤＮＡ 序列均是 ５＇端特异引物序列,表明是由５＇端特异引物单个引物扩增出来,全序列分析表明插入片段全长８３３ ｂｐ,是ＡＣＣ 氧化酶ｃＤＮＡ 基因编码区的一部分,５＇端缺少启始密码子ＡＴＧ,３＇端缺少部分编码序列和终止密码子ＴＡＡ⒚     

ACC氧化酶cDNA克隆及其对美洲黑杨体内乙烯产生的反义抑制

利用ＲＴ－ＰＣＲ技术克隆了番茄ＡＣＣ氧化酶基因ＬＥＥＴＨＹＢＲ编码区０．９ｋｂ的ｃＤＮＡ片段,经酶切图谱分析和序列分析鉴定后,反向插入到植物表达载体ｐＢｉｎ４３８中,构建了表达ＡＣＣ氧化酶反义ＲＮＡ的二元载体。用农杆菌侵染美洲黑杨叶片,在含卡那霉素的ＭＳ培养基上选择转化子和植株再生,通过ＰＣＲ检测筛选到１６株转基因杨树植株,Ｓｏｕｔｈｅｒｎｂｌｏｔ分析初步确证了外源基因是以单拷贝插入到杨树基因组中;对杨树幼苗乙烯释放量的测定结果表明转基因杨树的乙烯释放量为对照植株的２８％。     

Carbocyclic coformycin: a case study of the opportunities and pitfalls in the industrial search for new agrochemicals from nature

The work undertaken to isolate the novel, herbicidally active compound, carbocyclic coformycin, to obtain sufficient quantities of the compound for full biological evaluation, and to identify its biochemical mode of action is summarised. Although the compound was extremely active against some weed species, limitations in its spectrum of activity precluded further development. Carbocyclic coformycin exerts its biological action through a novel mode of action by the inhibition of the enzyme adenosine 5′-phosphate deaminase (EC 3.5.4.6) following phosphorylation in planta. From this work, the potential benefits of natural product research in the discovery of new agrochemicals are highlighted along with some of the possible pitfalls. © 1998 SCI.     

龙眼胚性愈伤组织ACC氧化酶基因的克隆及其在龙眼体胚发生过程中的表达分析

【目的】分离克隆龙眼(Dimocarpus longan Lour.)胚性愈伤组织乙烯合成关键酶ACO(1-aminocyclopropane-1-carboxylate oxidase)基因,并分析该基因在龙眼体细胞胚胎(以下简称龙眼体胚)发生过程中的表达情况。【方法】采用RT-PCR结合RACE法,获得龙眼胚性愈伤组织ACO基因的cDNA全长序列和DNA序列,运用生物信息学方法对序列进行分析,并通过实时荧光定量PCR(q-PCR)法研究该基因在龙眼体胚发生过程中的表达【。结果】克隆得到龙眼胚性愈伤组织ACO基因1315bp的cDNA全长序列(GenBank登录号为FJ534854),该cDNA的开放阅读框推定的氨基酸序列(含315个氨基酸)与其它植物ACO具有86%-47%同源性,包含了5'非编码区为86bp,3'非编码区为281bp,3'poly(A)尾长13bp;该基因的DNA序列(GenBank登录号为GU123929)长为1660bp,包含3个内含子,内含子的剪切位点均符合真核生物"GT-AG"规则;该基因在龙眼体胚各阶段均有表达,整个变化趋势呈字母"M"状。【结论】确定所获得的序列是龙眼胚性愈伤组织ACO基因的cDNA序列和DNA全长序列;该基因在不完全胚性紧实结构和心形胚的表达量为两个峰值。     

植物ACC合成酶在大肠杆菌中高效表达及表达产物活性测定

丝瓜ACC合成酶cDNA在T7启动子的作用下，经IPTG诱导，在大肠杆菌中高效表达，表达外源蛋白可占菌体总蛋白的45.2%.表达的ACC合成酶一部分以可溶的形式存在，具有与天然ACC合成酶相似的酶活性，将反应底物S-腺苷蛋氨酸(SAM)转化为1-氨基环丙烷-1-羧酸(ACC)并在细菌培养基中积累，另一部分则以无活性的包涵体形式存在于菌体中.     

大肠杆菌BL21(DE3)中表达重组蛋白的研究

为了研究在大肠杆菌BL21 (DE3)中重组蛋白的表达特点,探讨重组蛋白在大肠杆菌中表达时可溶性蛋白与包涵体出现的特点,从表达载体中表达了植物ACC合成酶.经SDS-PAGE电泳与双波长扫描分析,确定目的蛋白随表达时间、菌液密度的增加而增加.但是其最佳表达时间为2.5 h,菌液密度OD600为0.6,IPTG的最佳浓度为0.6 mmol/L,此时目的蛋白含量占全菌蛋白含量之比最高.植物ACC合成酶在大肠杆菌中表达时以包涵体的形式存在,未能检测到可溶性蛋白的表达.     

Chilling Tolerance of 10 Maize Genotypes as Related to Chilling-induced Changes in ACC and MACC Contents

We studied chilling-induced changes of 1-aminocydopropane-1-carboxylic acid (ACC) and of 1-(malonylamino) cyclopropane-1-carboxylic acid (MACC) contents in seedlings of ten maize genotypes with different chilling tolerance. Seedlings at the third leaf stage were chilled at 5°C and at 65% RH. Immediately before and after two and five days of chilling the contents of ACC and of MACC in the third leaf were measured. Water content and – after recovery – the degree of necrotic injuries and the percentage of seedling survival were also determined.
After 2 days of chilling, the ACC content increased in all genotypes investigated. The increase was significantly higher in the sensitive genotypes than in the tolerant ones. There was a significant correlation between ACC content and necrotic injuries of seedlings. Chilling for 5 days increased the ACC content further and the difference between the two groups of genotypes still existed.
The MACC content increased after 5 days of chilling in all genotypes investigated. The increase was greater in the tolerant genotypes than in the sensitive ones. However, the difference in MACC accumulation between the two groups of genotypes investigated was not significant, and thus no correlation between MACC accumulation and chilling susceptibility was found.
The possible causes for the increase of ACC and MACC contents under chilling conditions and the possibility of using the ACC content as an indicator of chilling tolerance in maize breeding are discussed.     

香石竹ACC氧化酶基因的克隆及其正义反义表达载体的构建

利用在Genebank上已报道的香石竹ACC氧化酶(ACO)基因的CDNA序列设计特异引物,以香石竹品种'MASTER'的eDNA为模板进行PCR扩增,克隆香石竹ACC氧化酶(AC0)基因.测序结果显示,克隆基因的序列与报道序列完全一致.将克隆的ACC氧化酶(AC0)基因分别连接到植物表达载体PBI121启动子CaMV 35S的上游及下游,构建香石竹ACC氧化酶(Aco)基因的正义表达栽体PBI121-ACO及反义表达载体PBI121-anti ACO.经PCR鉴定,基因已成功构建到表达裁体上.为香石竹抗衰老基因工程育种奠定了基础.     

新型饲料添加剂-ACC

ＡＣＣ是一种小分子的环状氨基酸,英文全名为：１－ａｍｉｎｏｃｙｃｌｏｐｒｏｐａｎｅ－１－ｃａｒｂｏｘｙｌｉｃ ａｃｉｄ,中文全名为１－氨基环丙烷－１－羧酸,是ＮＭＤＡ（Ｎ－甲基－Ｄ－天冬氨酸）的非士的宁敏感甘氨酸受体的部分强激动剂。ＡＣＣ通过作用ＮＭＤＡ受体而发挥其生理作用。目前已经发现ＡＣＣ具有改善动物繁殖性能、促进动物生长、抗神经中毒及保护大脑、神经、肾等作用。本文就ＡＣＣ近年来的研究进展、Ａ