杂草对除草剂非靶标抗性机理研究进展

随着除草剂的大面积持续使用,近年来抗性杂草种类增多,危害面积不断增加,危害程度逐渐加重。杂草对除草剂抗性问题业已成为威胁全球粮食安全的关键问题之一。杂草对除草剂的抗药机制主要分为靶标抗性和非靶标抗性,非靶标抗性主要包括对除草剂解毒能力增强、屏蔽作用或与作用位点的隔离作用等机理。本文主要对除草剂的非靶标抗性机制中的P450s、GSTs、ABC转运蛋白和谷胱甘肽转运体等进行综述,并对非靶标抗性机制研究前景进行展望。     

靶标酶在除草剂研究与开发中的作用

大多数除草剂都是通过特殊酶的抑制而产生杀草作用的。根据作用靶标对除草剂进行分类,了解靶标酶的作用机理及特性,对于新型除草剂的设计和杂草抗性的防治能够起到很大的帮助。本文介绍了谷胱甘肽转移酶(GST)、细胞色素P450酶、乙酰乳酸合成酶(ALS)、乙酞辅酶A羧化酶(ACCace)的研究进展。并分别从酶的生理功能、酶学特征、抑制剂作用机理、抑制剂的研究、抗性等方面进行了不同程度的阐述。     

柑橘大实蝇GSTd1基因的克隆、原核表达及重组蛋白GSTd1的酶学特征

为深入研究柑橘大实蝇Bactrocera minax嗅觉系统中气味信号降解机制，对柑橘大实蝇头部转录组进行分析，筛选谷胱甘肽S-转移酶(glutathione S-transferase,GST)基因，对其进行序列测定和分子生物学分析，利用实时荧光定量PCR技术检测该基因在柑橘大实蝇成虫各组织中的表达模式，并对该基因进行克隆和原核表达，采用Western blot对获得的编码蛋白GSTd1进行检测，并分析该蛋白的酶学特征。结果显示，成功获得1条具有完整开放阅读框的GST基因，将其命名为GSTd1，该基因完整开放阅读框为624 bp，编码207个氨基酸残基，预测蛋白分子量为23.72 kD，等电点为6.16，无信号肽，不含跨膜结构域，属于胞质型GST蛋白。序列比对和系统进化树分析结果表明柑橘大实蝇GSTd1属于昆虫特有的GST蛋白的Delta亚家族成员。柑橘大实蝇GSTd1基因在柑橘大实蝇成虫触角中的相对表达量最高。柑橘大实蝇GSTd1重组蛋白具有催化其通用底物1-氯-2,4-二硝基苯(1-chloro-2,4-dinitrobenzene,CDNB)的能力，最大反应速度和米氏常数分别为...     

杂草对9类常用不同作用机制除草剂的非靶标抗性机制研究进展

除草剂的应用为农业生产带来便利, 但长期、单一使用某一种或相同机制的除草剂也引发了杂草对除草剂的抗性问题。抗性杂草种类逐渐增加, 抗性形成机制复杂, 导致农田杂草的治理难度增加。杂草对除草剂的抗性机制主要分为两种, 一种是除草剂靶标位点基因的突变或过量表达导致的靶标抗性, 另一种是杂草对除草剂吸收、转运、固存和代谢等一个或多个生理过程发生变化导致的非靶标抗性。本文综述了杂草对9类不同作用方式除草剂的非靶标抗性机制的生理、生化和分子基础的研究进展, 以期为抗性杂草综合治理提供参考。     

杂草对AHAS抑制剂的抗药性分子机理研究进展

除草剂在田间的重复及不合理使用,导致了杂草抗药性的发生和发展。其中AHAS抑制剂由于靶标单一,抗性发展十分迅速。截至2009年,已有103种杂草对AHAS抑制剂产生了抗药性,占19类化学除草剂总抗药性杂草生物型的近1/3。从AHAS基因突变位点及种类与杂草抗药性水平的关系、AHAS基因突变与AHAS酶活性的关系、AHAS基因拷贝数与杂草抗药性的关系以及AHAS酶与除草剂结合前后的三维结构等方面,综述了杂草对AHAS抑制剂产生抗药性的机理,旨在为AHAS抑制剂抗性研究提供参考。并对自然种群目标基因的等位基因检测技术(ECOTILLING)和衍生型酶切扩增多态性序列(dCAPS)两种通过检测等位基因多态性的手段快速诊断抗药性杂草的新技术进行了介绍,讨论了延缓杂草抗药性发生和发展的策略。     

斜纹夜蛾SlGSTO2对拟除虫菊酯类和有机磷类杀虫剂体外代谢及其抗氧化活性

拟除虫菊酯类和有机磷类杀虫剂常用来防治包括斜纹夜蛾在内的多种鳞翅目害虫，目前斜纹夜蛾已对这两类杀虫剂产生了不同程度的抗性，谷胱甘肽S-转移酶 (glutathione S-transferases，GSTs) 广泛参与害虫对杀虫剂抗性的形成。前期通过转录组技术筛选到斜纹夜蛾SlGSTO2基因在拟除虫菊酯高抗种群中显著上调表达，为进一步探究该基因的功能，将其在大肠杆菌中异源表达，并测定重组蛋白对拟除虫菊酯类和有机磷类杀虫剂的代谢活性，同时采用抑菌圈试验测定了其抗氧化活性 。 结果表明:重组蛋白SlGSTO2仅对三氟氯氰菊酯有微弱的体外代谢活性，而对氰戊菊酯、高效氯氰菊酯、辛硫磷和毒死蜱均无代谢活性；抑菌圈试验结果表明，表达pET-26b(+)/SlGSTO2的大肠杆菌可以显著降低不同浓度过氧化氢异丙苯的抑菌圈直径，说明SlGSTO2具有抗氧化活性。研究结果丰富了对斜纹夜蛾GSTs Omega家族基因的功能认知。     

杂草抗药性及其治理策略研究进展

综述了杂草抗药性产生机理、杂草抗药性演化影响因素,并对其治理途径进行阐述,为杂草抗药性治理提供参考。除草剂抗药机制分为靶标抗性、非靶标抗性,其中靶标抗性包括除草剂作用位点改变、基因倍增及过量表达;非靶标抗性主要包括代谢解毒能力增强、屏蔽作用或与作用位点的隔离作用等。杂草抗药性演化受多种因素共同影响,不仅包括抗药性突变频率、除草剂选择压、杂草适合度及杂草种子库寿命四大因素,还与基因突变和遗传特征直接相关。在未来的杂草治理中,要经常进行田间杂草调查与鉴定,正确使用除草剂(交替使用、混用),并辅以合理的农艺管理措施来减缓杂草抗药性的演化速度。同时,应加强植物间化感作用的基础研究。     

吡啶甲酸类除草剂应用研究进展

吡啶甲酸类除草剂属合成激素类, 目前登记使用的有效成分包括氨氯吡啶酸、二氯吡啶酸和氯氨吡啶酸, 分别于1963年、1977年和2005年上市, 主要用于阔叶杂草和灌木的茎叶处理防控, 其中氯氨吡啶酸具有土壤封闭活性。吡啶甲酸类除草剂的作用靶标仍未明确, 有可能来自生长素结合蛋白家族。全世界报道的抗吡啶甲酸类除草剂杂草共涉及7种8个生物型。目前, 我国登记的除草剂品种中共有13个复配剂含氨氯吡啶酸和二氯吡啶酸, 1个含氨氯吡啶酸、氯氨吡啶酸和二氯吡啶酸;国外登记的吡啶甲酸类除草剂复配剂主要为与其他激素型除草剂、ALS(乙酰乳酸合酶)抑制剂、ACCase(乙酰辅酶A羧化酶)抑制剂的组合。该类除草剂仍然具有较好的应用前景, 在主要应用场景下常见杂草对氨氯吡啶酸、二氯吡啶酸、氯氨吡啶酸的敏感性仍需系统研究, 该类除草剂主要靶标杂草种群的抗药性水平也亟须检测。     

细胞色素P450酶系与植物的化学防御

简述了植物细胞色素P450酶系(P450s)在植物防御性物质的合成以及对除草剂等外源化学物质的解毒方面的功能,提出了利用分子生物学等技术,分离人们需要的P450基因,并在转基因植物中得到表达,培育出具有抗病虫、除草剂等功能的转基因作物的展望。     

miRNA在植物病毒基因组中的靶基因预测及分析

microRNA(miRNA)是一类长约22nt的内源性的非编码小RNA,在植物的生长发育和胁迫响应中起重要调控作用。本文通过生物信息学的方法,在miRBase中下载了12种植物的miRNA序列;在DPVweb和VIDE病毒数据库中查找侵染这些植物的病毒,并在NCBI数据库中搜索其基因组序列,共获得173种病毒的基因组序列。用psRNATarget在线软件预测植物miRNA在植物病毒中的靶基因,发现植物miRNA可能参与调控包括聚合蛋白、衣壳蛋白、逆转录酶、复制相关基因、通读蛋白、依赖RNA的RNA聚合酶等多种植物病毒基因的表达。     

Glutathione transferases and herbicide detoxification in suspension-cultured cells of giant foxtail (Setaria faberi)

Glutathione transferases (GSTs) catalysing the conjugation of 1-chloro-2,4-dinitrobenzene, the chloro-s-triazine herbicide atrazine, the chloroacetanilide herbicides metolachlor and alachlor and the diphenyl ether herbicide fluorodifen have been identified in suspension-cultured cells derived from the grass weed giant foxtail (Setaria faberi Herrm.). In contrast to suspension-cultured cells of maize, where atrazine-conjugating GSTs are lost during de-differentiation, the GSTs active toward this herbicide in S. faberi plants were also expressed in cultures, suggesting that these isoenzymes are subject to different regulation in the crop and weed. As a result, glutathione conjugation was the major route of atrazine metabolism in S. faberi cultures. Activities of these GSTs were maximal three days after sub-culturing when the cells were dividing most actively, when they were determined to be in the order CDNB>alachlor>metolachlor= fluorodifen>atrazine. This indicated that GSTs which are enhanced during cell division can metabolise herbicides. On the basis of activity per mg protein, GST activities in the cultures were between 20 and 60-fold higher than those determined in the foliage of S. faberi seedlings. The GSTs with activity towards CDNB were resolved into three peaks following anion-exchange chromatography at pH 7·8 using Q-Sepharose. Peak 1 GSTs were not retained, while peak 2 and peak 3 were sequentially resolved with an increasing concentration of salt. Peak 1 GSTs showed activity toward metolachlor and atrazine but showed little activity toward fluorodifen. Peak 2 and peak 3 GSTs were active toward atrazine and metolachlor, with peak 3 being particularly associated with activity toward fluorodifen. The GSTs in these peaks were then further purified using S-hexyl-glutathione-agarose affinity chromatography. In each case, the affinity-bound fraction of the GSTs consisted of 28 kDa and 26 kDa polypeptides, suggesting that the GST isoenzymes in S. faberi cultures are composed of related subunits. Our results demonstrate that the GST isoenzymes involved in herbicide metabolism in suspension cultures of a grass weed show a similar level of complexity to that determined in maize cell cultures. © 1998 SCI     

Elevated antioxidant response and induction of tau-class glutathione S-transferase after glyphosate treatment in Vigna radiata (L.) Wilczek

Glyphosate (N-(phosphonomethyl) glycine) is a broad-spectrum herbicide, acting on the shikimic acid pathway inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), thus obstructing the synthesis of tryptophan, phenylalanine, tyrosine and other secondary products. It has also been reported to generate oxidative stress which influences the antioxidant response of target plants. The effect of glyphosate application on total protein, CAT, POD and GST activities was investigated and elevated expression of the oxidative stress enzymes was obtained after glyphosate treatment.Tau-class GSTs are plant-specific, and are chiefly involved in xenobiotics and oxidative stress metabolisms. Many herbicides and safeners have been known to selectively induce tau-class GSTs in different plant species. Here we also report the induction of tau-class GSTs after glyphosate treatment in the seedling roots of two Vigna radiata varieties (PDM11 and PDM54). GSH-agarose affinity chromatography and mass spectrometry revealed that the tau-class GSTs induced in the two varieties were different; the tau-class GSTs present in the untreated controls were also different in the two varieties. The present study highlights the elevated antioxidant response, the induction of tau-class GST and the genotypic variation in the type of tau-GST in control and glyphosate treated varieties of V. radiata.     

Developmental changes in glutathione S‐transferase activity in herbicide‐resistant populations of Alopecurus myosuroides Huds (black‐grass) in the field

Herbicide‐resistant populations of Alopecurus myosuroides Huds (black‐grass) have become widespread throughout the UK since the early 1980s. Clear evidence suggests that more than one resistance mechanism exists, and glutathione S‐transferases (GSTs) have been implicated in resistance due to enhanced metabolism. This study reports the determination of GST activity in four UK black‐grass populations from field sites situated in the East Midlands. Data demonstrate that, as untreated plants in the field mature, there is an accompanying natural elevation of GST activity with natural environmental changes from winter to spring. We speculate that this endogenous change in enzyme activity with plant development in the field contributes to reduced efficacy of some graminicides applied in the spring. These observations are discussed in relation to predicting herbicide efficacy to achieve maximum control of this important grass weed. © 2001 Society of Chemical Industry     

Purification,characterization and comparison of glutathione S-transferases from black-grass (Alopecurus myosuroides Huds) biotypes

In the UK biotypes of black-grass (Alopecurus myosuroides Huds) showing resistance to both chlorotoluron (CTU) and aryloxyphenoxypropionate graminicides are increasingly being observed. Although the precise mechanisms involved in this resistance have yet to be identified, increased herbicide metabolism has been implicated as being involved in at least some cases of resistance. Glutathione S-transferases (GSTs) are a group of enzymes which have been demonstrated to metabolise herbicides in some plants, and the resistant black-grass biotype Peldon contains approximately double the GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) of susceptible biotypes. To investigate further the possible role of GSTs in herbicide resistance in black-grass, a purification procedure has been developed for these enzymes. A 27.5 kDa polypeptide possessing GST activity was purified from the susceptible biotype Herbiseed. Purification of GSTs from the resistant biotype Peldon also identified this polypeptide along with an additional 30 kDa polypeptide. An in-vitro kinetic study of both crude and purified GST extracts, and western blot analysis using antisera raised against the 27.5 kDa polypeptide, suggest that the 30 kDa polypeptide may possess GST activity, and is not a precursor of the 27.5 kDa polypeptide. These results are discussed and compared to GST profiles for other weeds and crops demonstrating herbicide resistance or tolerance. © 1999 Society of Chemical Industry     

Characterisation of Multiple Glutathione Transferases Containing the GST I Subunit with Activities toward Herbicide Substrates in Maize (Zea mays)

The glutathione transferases (GSTs) of maize with activities toward chloroacetanilide herbicides are relatively well characterised, but their range of substrate specificities has not been determined in detail. GST activities toward an extensive range of chemically diverse xenobiotic substrates, including the herbicides atrazine, alachlor, metolachlor and fluorodifen, have been determined in crude and purified preparations from the roots and shoots of dark-grown maize seedlings treated with and without the herbicide-safener dichlormid. With the exception of the activity toward atrazine, specific activities were higher in the roots than in the shoots in all cases. In untreated shoots activities were in the order atrazine=alachlor=metolachlor>fluorodifen with safener–treatment selectively increasing the activity toward the chloroacetanilides and fluorodifen. In the roots the highest GST activities toward herbicides were toward the chloroacetanilides. Dichlormid treatment resulted in an increase in activities toward all four herbicides in the roots of one maize cultivar (Pioneer 3394) but only enhanced the activities toward the chloroacetanilides and fluorodifen in cultivar Artus. Using the non-herbicide 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, anion-exchange chromatography showed that the roots and shoots contained a similar range of GST isoenzymes. All of these isoenzymes were enhanced in response to safeners, though the extent of this induction was organ-dependent. GST isoenzymes containing the GST I subunit were purified from safener-treated roots by a combination of hydrophobic interaction chromatography and affinity chromatography using Orange A agarose. Three isoenzymes could then be purified following resolution by anion-exchange chromatography. The three GSTs were termed GST I/I, GST I/II and GST I/III with GST I, II and III referring to the presence of 29 kDa, 27 kDa and 26 kDa subunits respectively. This revised nomenclature for the maize GSTs was considered necessary in view of the continued discovery of new isoenzymes, such as GST I/III, composed of subunits which have been previously described. GST I/I had measurable activity toward atrazine, low activities toward the other herbicides and appreciable activities toward a range of other xenobiotic substrates. GST I/II and the novel GST I/III isoenzymes both showed high activities toward the chloroacetanilides and fluorodifen but lower activities toward the other substrates and negligible activities toward atrazine. The GST II subunit of GST I/II also had activity as a glutathione peroxidase. Our results show that the GST I subunit can form dimers with the GST III subunit in addition to the GST I and GST II subunits and that the degree of specificity toward herbicide substrates of the respective isoenzymes is greater than previously reported. Our results also suggest that the safener-inducible GST II subunit has additional activities as a glutathione peroxidase. © 1997 SCI.     

Purification and partial characterization of a glutathione S-transferase from the two-spotted spider mite, Tetranychus urticae

Glutathione S-transferases (GSTs) are known to catalyze conjugations by facilitating the nucleophilic attack of the sulfhydryl group of endogenous reduced glutathione on electrophilic centers of a vast range of xenobiotic compounds, including insecticides and acaricides. Elevated levels of GSTs in the two-spotted spider mite, Tetranychus urticae Koch, have recently been associated with resistance to acaricides such as abamectin [Pestic. Biochem. Physiol. 72 (2002) 111]. GSTs from acaricide susceptible and resistant strains of T. urticae were purified by glutathione-agarose affinity chromatography and characterized by their Michaelis-Menten kinetics towards artificial substrates, i.e., 1-chloro-2,4-dinitrobenzene and monochlorobimane. The inhibitory potential of azocyclotin, dicumarol, and plumbagin was low (IC₅₀ values > 100 μM), whereas ethacrynic acid was much more effective, exhibiting an IC₅₀ value of 4.5 μM. GST activity is highest in 2-4-day-old female adults and dropped considerably with progressing age. Furthermore, molecular characteristics were determined for the first time of a GST from T. urticae, such as molecular weight (SDS-PAGE) and N-terminal amino acid sequencing (Edman degradation). Glutathione-agarose affinity purified GST from T. urticae strain WI has a molecular weight of 22.1 kDa. N-terminal amino acid sequencing revealed a homogeneity of ≈50% to insect GSTs closely related to insect class I GSTs (similar to mammalian Delta class GSTs).     

Co-induction of glutathione-S-transferases and multidrug resistance associated protein by xenobiotics in wheat

Herbicide safeners are known to protect monocotyledonous crops from herbicide injury by accelerating the metabolism of herbicides. We have investigated the effects of the safener cloquintocetmexyl, which protects small-grain cereals against the graminicidal herbicide, clodinafop-propargyl. Subtractive suppression hybridisation was used to identify wheat genes which are up-regulated by treatment not only with cloquintocet-mexyl but also with phenobarbital, which is known to stimulate xenobiotic metabolism in animals and plants. DNA sequences of five glutathione transferases (GSTs) belonging to three different classes and a multidrug resistance associated protein (MRP) homologue were identified in the screen. The chemical inducibility of these clones was confirmed by Northern analysis. The MRP protein was shown to be induced by treatments with cloquintocet-mexyl and phenobarbital and to be localised to the tonoplast. Since clodinafop-propargyl is not known to be metabolised by glutathionylation, the significance of GST induction is interpreted in terms of a generalised response to chemical stress, particularly the generation of active oxygen species. This work establishes herbicide safeners as useful tools for the identification of genes encoding herbicide-metabolising enzymes.     

Glutathione transferases in herbicide-resistant and herbicide-susceptible black-grass (Alopecurus myosuroides)

Glutathione transferase (GST) activities toward the selective herbicide fenoxaprop-ethyl, together with thiol contents, have been compared in seedlings of wheat (Triticum aestivum) and two populations of black-grass (Alopecurus myosuroides) which are resistant to a range of herbicides (Peldon and Lincs E1), and a black-grass population which is susceptible to herbicides (Rothamsted). GST activities toward the non-cereal herbicides metolachlor and fluorodifen were also determined. On the basis of enzyme specific activity, GST activities toward fenoxaprop-ethyl in the leaves were in the order wheat>Peldon=Lincs E1>Rothamsted, while with fluorodifen and metolachlor the order was Peldon=Lincs E1>Rothamsted>wheat. Using an antibody raised to the major GST from wheat, which is composed of 25-kDa subunits, it was shown that the enhanced GST activities in both Peldon and Lincs E1 correlated with an increased expression of a 25-kDa polypeptide and the appearance of novel 27-kDa and 28-kDa polypeptides. Leaves of both wheat and black-grass contained glutathione and hydroxymethylglutathione, with the concentrations of glutathione being in the order Peldon>Lincs E1=Rothamsted=wheat. However, in glasshouse dose-response assays, the Lincs E1 population showed much greater resistance to fenoxaprop-ethyl than Peldon. We conclude that high GST activities and the availability of glutathione may contribute partially to the relative tolerance of black-grass to herbicides detoxified by glutathione conjugation. Although herbicide-resistant populations show enhanced GST expression, in the case of fenoxaprop-ethyl the associated increased detoxifying activities alone cannot explain the differences between populations in the degree of resistance seen at the whole plant level. ©1997 SCI     

Biochemical effects of acute phoxim administration on antioxidant system and acetylcholinesterase in Oxya chinensis (Thunberg) (Orthoptera: Acrididae)

The study was undertaken to evaluate the effects of different concentrations of phoxim on acetylcholinesterase (AChE) and esterase (EST) activities, and antioxidant system after topical application to Oxya chinensis. The results showed that phoxim inhibited AChE activity, and did not cause significant changes in the EST activity and the levels of malondialdehyde (MDA) and reduced glutathione (GSH). After phoxim administration, superoxide (SOD) and catalase (CAT) activities showed a biphasic response with an initial increase followed by a decline in their activities. Glutathione reductase (GR) and glutathione peroxidase (GPx) activities were inhibited in comparison with the control. Glutathione S-transferase (GST) activity showed irregular changes. Its activity increased significantly at the concentrations of 0.06 and 0.12 μg/μL and decreased at the concentrations of 0.09 and 0.24 μg/μL compared with the control. Changes in SOD, CAT, GST, GPx, and GR activities indicated that phoxim caused oxidative damage in O. chinensis. However, no significant changes in MDA content suggested that these enzymes played important roles in scavenging the oxidative free radicals induced by phoxim in O. chinensis. The formation of oxygen free radicals might be a factor in the toxicity of phoxim.