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耐草甘膦菜豆耐性机理的初步研究 总被引：2，自引：0，他引：2
采用液谱测定耐性、感性菜豆叶片对草甘膦的吸收及草甘膦传导入根中的量。耐性、感性菜豆吸收、传导草甘膦无差异。耐性、感性菜豆 EPSP合成酶提取物中的蛋白质含量分别为 3.0 0 mg/ m L和 3.0 8mg/ m L ,EPSP合成酶的比活性分别为 2 .13nmol· min-1· mg-1蛋白和 1.97nmol· min-1· mg-1蛋白 ,但耐性、感性菜豆 EPSP合成酶比活性被草甘膦不同浓度抑制的差异大 ,抑制耐性菜豆 EPSP合成酶活性的草甘膦浓度 I50 为 19.2μmol/ L ,而感性的 I50 为 6 .3μmol/ L。两种菜豆对草甘膦的耐性差异在于各自的 EPSP合成酶比活性被草甘膦的抑制程度不同。 相似文献
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感性、耐性菜豆从萌芽到24d的EPSP合成酶比活性是不同的,但变化规律相似。出苗时比活性较高,10d时降至最低点,随后上升,16d时达到最高点,然后缓慢下降;两种菜豆在生长的同一天酶比活性差异小,最大倍数仅为1.1;用草甘膦处理两种三出复叶时的菜豆1d后酶比活性上升15%～25%。实验测定了经4步纯化,比活性达6219.4nmol· min-1· mg-1蛋白以上的两种菜豆EPSP合成酶的一些酶学性质。PEP是酶的最适底物,感性、耐性 Km(PEP)分别为3.5和7.1μmol· L-1,两者相差2倍,亲和力Ki(草甘膦)分别是6.2和 16.7μmol· L-1,两者相差2.7倍。两种菜豆对草甘膦耐性不同的酶学机理在于耐性菜豆的EPSP合成酶与草甘膦的亲和力小,与酶底物亲和力大;而感性菜豆EPSP酶则与草甘膦的亲和力大,与其底物亲和力小。 相似文献
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EPSP合成酶 ( 5 -烯醇丙酮酸莽草酸 - 3-磷酸合酶 )是除草剂靶标酶之一 [1 ] ,也是抗草甘膦转基因作物的关键性酶 [2 ] ,它催化一分子的莽草酸 - 3-磷酸 ( S3P)和烯醇式丙酮酸 ( PEP)成EPSP,从而导致芳香族氨基酸——色氨酸、酪氨酸、苯丙氨酸的生物合成。 EPSP合成酶存在于细菌、真菌和植物中 ,但由于其性质的不稳定性、不均匀性和低丰度 ,造成分离纯化困难 [3] 。作者以菜豆 ( Phaselusvulgaris L .)幼苗为原料 ,经快速纯化 (整个纯化过程少于 1 .5 h)获得的EPSP合成酶产品能用于除草剂的筛选工作 ,为以 EPSP合成酶为靶标的生… 相似文献
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用耐性菜豆根尖为材料，以λgt10为载体获得了4.8×105个重组噬菌体的cDNA文库。以植物EPSP合成酶基因保守序列合成二段探针，经噬菌体原位杂交筛选出了阳性克隆，并进行了酶切鉴定。测定 cDNA 序列长度为2024个核苷酸，其中编码长1569个核苷酸，共编码523个氨基酸和一个终止密码子，与已发表的其它植物成熟EPSP合成酶氨基酸序列相比具有很高的同源性（≥84.8%），但进入叶绿体运输肽的氨基酸同源性低。以耐性菜豆EPSP合成酶的cDNA序列为模板，用RT-PCR方法扩增感性菜豆EPSP合成酶cDNA片段，并克隆于pUC18质粒上，经测定序列并比较发现:感性菜豆EPSP合成酶cDNA核苷酸序列1737位碱基为G，而耐性的为C，从而表达出的513位氨基酸残基感性的为E，耐性的为Q，表明两种菜豆EPSP合成酶cDNA核苷酸序列一位点的差异是它们对草甘膦耐性不同的原因。 相似文献
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<正>1草甘膦抗性现状及面临的其他问题草甘膦为内吸传导型慢性广谱灭生性除草剂,主要抑制植物物体内烯醇丙酮基莽草素磷酸合成酶(EPSP),从而抑制莽草素向苯丙氨酸、酪氨酸及色氨酸的转化,使蛋白质的合成受到干扰,从而导致植物死亡。由于草甘膦优异的杀草活性、广泛的杀草谱、较低的土壤残留、较长的控草时间,加上抗除草剂转基因作物的广泛种植,使其成为全球销量第一的除草剂品种。然而由于长时间大量单一连续使用草甘膦,杂草的抗性问题已经非常突出。到目前已经公布了有31种 相似文献
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为了克隆抗草甘膦基因并在原核表达系统中分析其抗性水平,利用200 μmol/L草甘膦平板从草甘膦严重污染的土壤中筛选到1株抗草甘膦菌株G6PP,经电镜和16S rDNA鉴定为恶臭假单胞菌;以该菌株基因组DNA为模板,通过PCR方法扩增出5-烯醇丙酮莽草酸-3-磷酸酯合成酶(EPSPS)基因,该基因编码440个氨基酸,亚克隆到原核表达载体pEET-28a中构建原核表达载体pET-G6;将重组表达载体转化大肠杆菌BL21 (DE3)中,经IPTG诱导,表达出46 ku的融合蛋白;携带pET-G6质粒的大肠杆菌BL21 (DE3)在液体M63培养基中能耐受150 μmol/L草甘膦的抑制.本研究结果表明克隆到的G6基因具有一定的抗草甘膦活性,对抗草甘膦作物的培育具有实践意义. 相似文献
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为明确广东省稻菜轮作区中牛筋草对10种常用除草剂的抗性水平及抗性分子机制,采用整株生物测定法测定广东省稻菜轮作区内8个牛筋草种群P1～P8对草甘膦、草铵膦和乙酰辅酶A羧化酶(acetyl-CoA carboxylase,ACCase)抑制剂类等10种除草剂的抗性水平,并进一步分析P1和P8种群相关靶标酶基因5-烯醇丙酮酰莽草酸-3-磷酸合酶(5-enolpyruvyl-shikimate-3-phosphate synthase,EPSPS)、谷氨酰胺合成酶(glutamine synthetase,GS)和ACCase的部分功能区序列特征。结果显示,牛筋草P1～P8种群对草甘膦抗性指数为敏感种群的5.9倍～17.7倍,其中P8种群对草甘膦的抗性水平最高;8个种群对草铵膦也产生了不同程度的抗性,抗性指数为敏感种群的2.3倍～14.2倍,其中P1种群抗性最高。牛筋草P1和P8种群均对ACCase抑制剂类除草剂精喹禾灵、氰氟草酯和噁唑酰草胺产生了交互抗性;P1种群ACCase基因在第2 041位氨基酸处发生突变,该突变在牛筋草种群中首次发现;而P8种群ACCase基因则在第2 027位氨基... 相似文献
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高锰胁迫下草甘膦对空心莲子草体内莽草酸含量和抗氧化酶活性的影响 总被引：2，自引：0，他引：2
鉴于高锰胁迫下空心莲子草Alternanthera philoxeroides对草甘膦的耐药性增强,在水培条件下研究了不同浓度锰条件下草甘膦处理后该草体内莽草酸的积累和主要抗氧化酶系统的响应。次高浓度锰(0.31 mmol/L)条件下培养120 d后空心莲子草体内过氧化氢酶(CAT)活性显著高于常规浓度锰处理(0.009 1 mmol/L,对照);高浓度锰(2.45 mmol/L)条件下超氧化物歧化酶(SOD)活性升高,CAT活性下降。草甘膦(按草甘膦酸68 g/hm2)茎叶处理后6 d内,常规锰浓度培养的空心莲子草体内莽草酸含量比用草甘膦刚处理时增加了31.9％~226.0%,且显著高于同一时间次高锰和高锰的处理;不同锰浓度下培养的空心莲子草体内过氧化物酶(POD)和CAT、SOD活性均为先升高后再逐渐下降,但次高锰处理的该3种酶活性均高于对照,高锰处理的SOD和POD活性高于对照,而CAT活性与对照相当。上述结果表明,在较高锰浓度下空心莲子草能启动抗氧化酶系统而能有效地清除自由基;在草甘膦处理后初期,高锰条件下空心莲子草体内莽草酸途径受抑制程度较轻,抗氧化酶活性较高,这可能是空心莲子草耐高锰和高锰条件下该草耐草甘膦的部分机制。 相似文献
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Three cell lines of groundnut (Arachis hypogaea L), an important oilseed legume, were selected on glyphosate using in-vitro culture techniques. The cell lines isolated through single as well as stepwise selection procedures showed c 20-fold increase in glyphosate tolerance as compared to the unselected control cell line. Studies on the biochemical mechanism of glyphosate tolerance in these cell lines showed a significant increase in the total extractable activity of the target enzyme, 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), which was further confirmed with immunological data. The over-expressed EPSP synthase activity was, however, subject to inhibition by glyphosate in vitro. Two other key regulated enzymes of the shikimic acid pathway, 3-deoxy-D -arabino heptulosonate 7-phosphate (DAHP) synthase (EC 4.1.2.15) and chorismate mutase (CM) (EC 5.4.99.5) did not show any change in specific activity in the selected cell lines. The enhanced activity of EPSP synthase in the tolerant cell lines was found to be stably inherited in the absence of selection pressure. © 1999 Society of Chemical Industry 相似文献
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Gene polymorphisms in glyphosate-resistant and -susceptible biotypes of Eleusine indica from Malaysia 总被引：3，自引：0，他引：3
Summary Resistant (R) and susceptible (S) biotypes of Eleusine indica were collected from four areas, namely Chaah, Lenggeng, Bidor and Temerloh, in Malaysia. Restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR)-RFLP analyses using Sph I restriction enzyme were able to differentiate the R biotype from the S biotype by showing R-specific and S-specific polymorphisms in E. indica from three of the areas, with the exception of Temerloh where no polymorphisms were detected. The different DNA profiles for the R biotypes obtained indicate that Sph I is not a useful diagnostic marker. The DNA polymorphisms detected in the 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase gene suggest that there are different mutation events leading to development of resistance to glyphosate. Partial sequencing of the EPSP synthase gene confirmed different mutations occurring with substitution of proline with serine or threonine at amino acid 106 for the R biotype in Chaah, Bidor and Temerloh. 相似文献
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Vande Berg BJ Hammer PE Chun BL Schouten LC Carr B Guo R Peters C Hinson TK Beilinson V Shekita A Deter R Chen Z Samoylov V Bryant CT Stauffer ME Eberle T Moellenbeck DJ Carozzi NB Koziel MG Duck NB 《Pest management science》2008,64(4):340-345
BACKGROUND: Glyphosate tolerance is a dominant trait in modern biotech crops. RESULTS: A gene encoding a glyphosate-tolerant EPSP synthase (aroA(1398)) from bacterial strain ATX1398 was cloned and characterized. The protein is initiated at a GTG translational start codon to produce a protein that provides robust glyphosate resistance in Escherichia coli (Mig) Cast & Chalm. The aroA(1398) protein was expressed and purified from E. coli, and key kinetic values were determined (K(i) = 161 microM; K(m)(PEP) = 11.3 microM; k(cat) = 28.3 s(-1)). The full-length enzyme is 800-fold more resistant to glyphosate than the maize EPSP synthase while retaining high affinity for the substrate phosphoenol pyruvate. To evaluate further the potential of aroA(1398), transgenic maize events expressing the aroA(1398) protein were generated. T(0) plants were screened for tolerance to glyphosate sprays at 1.3x commercial spray rates, and T(1) plants were selected that completely resisted glyphosate sprays at 1x, 2x and 4x recommended spray rates in field trials. CONCLUSION: These data suggest that aroA(1398) is a suitable candidate for conferring glyphosate tolerance in transgenic crop plants. 相似文献
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Laurence Arnaud Franoise Nurit Patrick Ravanel Michel Tissut 《Pest management science》1994,40(3):217-223
Enolpyruvylshikimate phosphate (EPSP) synthase activity has been measured in different organs of 1–14-day-old wheat seedlings growing in culture chambers. Enzyme activity was first detected after two days, and reached its maximum value at day 4. It remained almost unchanged for 14 days thereafter. During germination, one of the most important sources of enzyme was the seed scutellum. Later, the developed leaves contained 43% of the whole stock of enzyme. Although the enzyme distribution was not identical under light or in the dark, there was no evident light induction of EPSP synthase. High enzyme activity was found in the coleoptile, continuing for the whole life-time of this organ, and which might be associated with its lignification. Five to 40% of the total enzyme was found in the roots, reaching a maximum at 14 days. At this stage, glyphosate applied to the first grown leaf was phloem-transported to the sink organs, with no clear relationship being shown between EPSP synthase activity and herbicide quantity in each organ. At day 4, glyphosate treatment, by soaking the seedlings in the herbicide solution, was followed by an active phloem transport from the scutellum to the growing parts. The average internal concentration of radiolabelled compounds (glyphosate plus its possible derivatives) which induced wheat seedling growth decrease, was shown to reach 50 to 100 μM under our conditions. 相似文献
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Debrah F Lorraine-Colwill Tim R Hawkes Patricia H Williams Simon AJ Warner Peter B Sutton Stephen B Powles Christopher Preston 《Pest management science》1999,55(4):489-491
Annual ryegrass (Lolium rigidum) is a widespread and important weed of Australia and populations of this weed have developed resistance to most major herbicides, including glyphosate. The possible mechanisms of resistance have been examined in one glyphosate-resistant Lolium population. No major differences were observed between resistant and susceptible biotypes in respect of (i) the target enzyme (EPSP synthase), (ii) DAHP synthase, the first enzyme of the target (shikimate) pathway, (iii) absorption of glyphosate, or (iv) translocation. Following treatment with glyphosate, there was greater accumulation of shikimate (derived from shikimate-3-Pi) in susceptible than in resistant plants. In addition, the resistant population exhibited cross-resistance to 2-hydroxy-3-(1,2,4-triazol-1-yl)propyl phosphonate, a herbicide which, although structurally similar to glyphosate, acts at an unrelated target site. On the basis of these observations we speculate that movement of glyphosate to its site of action in the plastid is involved in the resistance mechanism. © 1999 Society of Chemical Industry 相似文献
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采用盆栽土壤基施0、15、30 kg·hm-2三个氮素水平试验,研究了芸豆荚果形成后不同氮素水平下叶片和荚果的碳、氮代谢主要酶活性以及抗氧化酶系活性的变化。结果表明,芸豆荚果形成后,随着土壤供氮水平的增加,荚果的磷酸烯醇式丙酮酸羧化酶活性、多酚氧化酶活性、超氧化物歧化酶和过氧化氢酶活性逐渐增强;叶片的蔗糖合成酶活性、蔗糖磷酸合成酶活性、硝酸还原酶和磷酸烯醇式丙酮酸羧化酶活性大于同一供氮水平荚果的活性;且荚果的总可溶性糖和可溶性蛋白含量高于叶片;叶片的磷酸烯醇式丙酮酸羧化酶的活性与荚果的蔗糖磷酸合成酶和硝酸还原酶活性呈显著相关性。由此可以得出,源器官是芸豆植株的代谢活跃中心,而库器官成为植株的生长中心,且后者的碳、氮代谢过程主要受前者的调控。 相似文献