Trp_(548)Met mutation of acetolactate synthase in rice confers resistance to a broad spectrum of ALS-inhibiting herbicides

Herbicide resistance in crop plants is valuable for integrated weed management in agriculture. Herbicide resistant rice, in particular, is important to management of weedy rice, a close relative of cultivated rice and a noxious weed prevalent in rice fields that remains challenging to farmers worldwide. Herbicide resistant plants can be obtained through transgenic approach or by mutagenesis of regular plant and screening of mutants with elevated resistance to herbicide. In this study, we conducted ethyl methyl sulfonate mutagenesis(EMS) to elite indica cultivar Huanghuazhan(HHZ) and screened for mutants resistant to imazapic, a herbicide that can inhibit the acetolactate synthase(ALS) in plants. We obtained three mutants of Os ALS gene that have not been reported previously in rice. One of the mutants, with Trp_(548) changed to Met(W_(548)M), was analyzed in more details in this study. This mutation had no negative effect on the plant physiology and morphology as well as rice yield. Compared with the imidazolinone-resistant mutant S_(627)N(Ser_(627) changed to Asn) that has been deployed for Clearfield rice development, W_(548)M mutant showed high levels of resistance to a broad spectrum of five families of ALSinhibiting herbicides, in addition to a higher level of resistance to herbicides of the imidazolinone family.The herbicide-resistance was stably inherited by crossing into other rice lines. Thus, the W_(548)M mutation provides a valuable resource for breeding of herbicide resistant rice and weed management.

Tissue-specific Expression of Acetolactate Synthase (ALS), Male Sterility-inducing Effect of Tribenuron-methyl and Its Effect on ALS Activity in Brassica napus L.

[Objectives]This study was conducted to provide a basis for the rapid identification of the drug spraying effect in early stage and the molecular mechanism of chemical hybridizing in Brassica napus L.[Methods]Quantitative RT-PCR analysis showed that ALS was constitutively expressed in various tissues of 096030,including flower buds,four floral organs (calyxes,petals,stamens and pistils),roots,stems and leaves.ALS was prominently expressed in leaves and was expressed weakly in the petals and stamens.The male sterility-inducing effects of tribenuron-methyl on such two Brassica napus L.varieties as Ningyou18 and 096030 were investigated.[Results]Plants were twice sprayed with 0.2 μg/ml tribenuron-methyl on leaves.The results showed that 8-10 ml of tribenuron- methyl was applied per plant for the first time at bolting stage with 1-2 mm flower buds on 15-20 cm inflorescence,and the second spray was performed with 8-10 ml of tribenuron-methyl per plant 10 d later.The results showed that the percentage of the full sterile plants reached 100%,which lasted for the whole flowering period,and the relative seed setting rate was only about 4%.Thus,this method could fullfill the requirement of hybrid seed production in field.The in-vivo enzyme activity of acetolactate synthase (ALS) was assayed using 2 mm buds collected 3 d after spray.The results showed that 0.2 μg/ml tribenuron-methyl inhibited ALS activity.The ALS activity of Ningyou 18 (CK) and Ningyou 18 (0.2 μg/ml) was 3.20 and 1.30 μmol/(mg·h),respectively,and the ALS activity of 096030 (CK) and 096030 (0.2 μg/ml) was 3.37 and 1.25 μmol/(mg·h),respectively.The relative enzyme activity of ALS in Ningyou18 and 096030 was 40.63% and 37.23%,respectively,both of which decreased significantly.[Conclusions]These results showed that the change of ALS activity may be used as an index for quickly identifying and predicting the chemical hybridizing effect of tribenuron-methyl.

A nonsynonymous mutation in an acetolactate synthase gene (Gh_D10G1253) is required for tolerance to imidazolinone herbicides in cotton

Background Herbicide tolerance in crops enables them to survive when lethal doses of herbicides are applied to surrounding weeds.Herbicide-tolerant crops can be developed through transgenic approaches or traditional mutagenesis approaches.At present,no transgenic herbicide tolerant cotton have been commercialized in China due to the genetically-modified organism(GMO)regulation law.We aim to develop a non-transgenic herbicide-tolerant cotton through ethyl methanesulfonate(EMS)mutagenesis,offering an alternative choice for weed management.Results Seeds of an elite cotton cultivar Lumianyan 37(Lu37)were treated with EMS,and a mutant Lu37-1 showed strong tolerance to imidazolinone(IMI)herbicides was identified.A novel nonsynonymous substitution mutation Ser642Asn at acetolactate synthase(ALS)(Gh_D10G1253)in Lu37-1 mutant line was found to be the potential cause to the IMI herbicides tolerance in cotton.The Ser642Asn mutation in ALS did not present among the genomes of natural Gossypium species.Cleaved amplified polymorphic sequence(CAPS)markers were developed to identify the ALS mutant allele.The Arabidopsis overexpressing the mutanted ALS also showed high tolerance to IMI herbicides.Conclusion The nonsynonymous substitution mutation Ser642Asn of the ALS gene Gh_D10G1253 is a novel identi-fied mutation in cotton.This substitution mutation has also been identified in the orthologous ALS genes in other crops.This mutant ALS allele can be used to develop IMI herbicide-tolerant crops via a non-transgenic or transgenic approach.

Precise base editing of non-allelic acetolactate synthase genes confers sulfonylurea herbicide resistance in maize

Single-nucleotide polymorphisms contribute to phenotypic diversity in maize. Creation and functional annotation of point mutations has been limited by the low efficiency of conventional methods based on random mutation. An efficient tool for generating targeted single-base mutations is desirable for both functional genomics and precise genetic improvement. The objective of this study was to test the efficiency of targeted C-to-T base editing of two non-allelic acetolactate synthase(ALS) in generating sulfonylurea herbicide-resistant mutants. A CRISPR/Cas9 nickase-cytidine deaminase fused with uracil DNA glycosylase inhibitor(UGI) was employed to achieve targeted conversion of cytosine to thymine in ZmALS1 and ZmALS2. Both protoplasts and recovered mutant plants showed the activity of the cytosine base editor, with an in vivo efficiency of up to 13.8%. Transgene-free edited plants harboring a homozygous ZmALS1 mutation or a ZmALS1 and ZmALS2 double mutation were tested for their resistance at a dose of up to 15-fold the recommended limit of chlorsulfuron, a sulfonylurea herbicide widely used in agriculture. Targeted base editing of C-to-T per se and a phenotype verified in the generated mutants demonstrates the power of base editing in precise maize breeding.

水稻乙酰乳酸合酶Ala179Val突变赋予ALS抑制剂类除草剂广谱抗性

为了鉴定新型水稻突变体ALS179对乙酰乳酸合酶(ALS,acetolactate synthase)抑制剂类除草剂的抗性,本研究以野生型水稻华航31(HH31)、耐咪唑啉酮除草剂水稻ALS627突变体和甲基磺酸乙酯(EMS,ethyl methyl sulfone)诱变的新型水稻突变体ALS179为试验材料,通过不同浓度下的4类乙酰乳酸合酶抑制剂类除草剂包衣浸种和苗期喷施处理,进一步测定表型及相关酶活性指标来探究突变体ALS179的抗性。结果表明,经过除草剂包衣浸种以及苗期喷施处理后,突变体ALS179对苯磺隆、咪唑乙烟酸、双草醚及啶磺草胺具有不同程度的抗性,且乙酰乳酸合酶、过氧化物酶、过氧化氢酶和超氧化物歧化酶的活性随除草剂浓度的升高呈下降趋势。除了20×,30×的咪唑乙烟酸处理条件下过氧化氢酶和过氧化物酶的酶活性低于野生型HH31外,其他处理条件下ALS179的乙酰乳酸合酶、超氧化物歧化酶、过氧化氢酶和过氧化物酶的酶活性均高于野生型HH31。因此,本研究发现Ala179Val突变赋予了对ALS抑制剂类除草剂的广谱抗性,为后续ALS类除草剂广谱抗性水稻品系的培育提供遗传种质资源。

抗ALS抑制剂类除草剂分子标记的开发及应用

杂草严重制约水稻生产,培育具有乙酰乳酸合成酶(acetolactate synthase,ALS)抑制剂的广谱抗除草剂水稻新品种是应对稻田杂草危害的有效途径之一。本研究鉴定了抗ALS抑制剂类除草剂金粳818与感除草剂日本晴ALS基因的差异位点,开发特异分子标记。对以携带ALS抗性基因型的金粳818、津稻372为亲本的杂交后代进行除草剂喷施和分子标记辅助选择。开发了818ALSF/R、HDALSF/R两对分子标记,可以快速区分纯合显性、杂合、纯合隐性3种基因型,且与除草剂喷施结果一致;选育得到22S9和22S4两份抗除草剂优异水稻种质资源材料。通过对杂交后代分子标记辅助选择可快速筛选出具有ALS除草剂抗性基因的种质资源材料,提高了在抗ALS抑制剂类除草剂水稻种质中的筛选效率。

Activity of Acetolactate Synthase from Maize (Zea mays L.) as Influenced by Chlorsulfuron and Tribenuron-methyl

Study on relative sensitivity of maize (Zea mays L.) Nongda108 and Nongda3138 to sulfony-lurea herbicide chlorsulfuron and tribenuron-methyl using maize taproot length by sand bioassy indicated that, Nongda3138 had higher tolerance to chlorsulfuron and tribenuron-methyl than Nongda108 did. Chlorsulfuron had stronger growth inhibition to maize Nongda108 and Nongda3138 than tribenuron-methyl did. Study on target enzyme of sulfonylurea herbicide acetolactate synthase (ALS) showed that, chlorsulfuron and tribenuron-methyl inhibited ALS in vitro strongly, and non-competitively. In the same concentration of inhibitors, chlorsulfuron had stronger ALS activity inhibition than tribenuron-methyl did. Lower level of chlorsulfuron and tribenuron-methyl has no ALS activity inhibition in vivo, the ALS inhibition only occurred in the condition of high concentration of chlorsulfuron and tribenuron-methyl in vivo.

The Mutated Acetolactate Synthase Gene from Rice as a Non-Antibiotic Selection Marker for Transformation of Bamboo Cells

Previously, we developed a particle bombardment-mediated transformation protocol in Phyllostachys nigra bamboo by expressing hygromycin phosphotransferase gene (HPT) and neomycin phosphotransferase II gene (NPT II). Although these marker genes could introduce to several tissue cultured organs (e.g. leaves, buds, and calli) of Phyllostachs bamboo species, some organs showed a high susceptibility and/or a low selectivity to hygromycin and kanamycin. In this report, therefore, we describe advantages and technical details for generating stable transgenic bamboo cells using the particle bombardment method with the mutated-acetolactate synthase gene (mALS) from rice (W548L/S627IOsALS) as a non-antibiotic selection marker. A facile and efficient transformation was achieved with the mALS gene and enhanced fluorescent protein gene (mCherry). Approximately 490 and 1400 mCherry-expressing cells/dish/shot in average were observed in both P. bambusoides and P. nigra under fluorescent stereo-microscope. Stable transgenic bamboo cell lines were generated in a selection medium supplemented with 0.1 μM of bispyribac-sodium (BS) as ALS inhibitor. The integration of mALS gene was identified by in vivo ALS enzyme assay and a PCR-restriction fragment length polymerphism (RFLP) based detection procedures.

Herbicide-Resistant Mutations in Acetolactate Synthase Can Reduce Feedback Inhibition and Lead to Accumulation of Branched-Chain Amino Acids

The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids that are critical for animal growth and development. Animals need to obtain BCAAs from their diet because they cannot synthesize them. Plants are the ultimate source of these amino acids. Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of BCAAs. The metabolic control of BCAA biosynthesis involves allosteric regulation of ALS by the end-products of the pathway, i.e., valine, leucine and isoleucine. ALS holoenzyme seems to consist of two large catalytic subunits and two small regulatory subunits. In a previous study, using homologous recombination dependent gene targeting we created rice plants in which W548Land S627I mutations were induced into the endogenous gene encoding the ALS catalytic subunit. These two amino acid substitutions conferred hypertolerance to the ALS-inhibiting herbicide bispyripac-sodium. In this study, we revealed that feedback regulation by valine and leucine was reduced by these two amino acid substitutions. Furthermore, in leaves and seeds of ALS mutants with W548Land/or S627I substitution, a 2- to 3-fold increase in BCAAs was detected. Our results suggest that the ALS catalytic subunit is also involved in feedback regulation of ALS, and that judicious modification of the regulatory and catalytic subunits of ALS-coding genes by gene targeting can lead to the efficient accumulation of BCAA in plants.

水稻抗咪唑啉酮类除草剂基因ALS功能标记的开发与应用

选育和利用抗除草剂水稻品种具有重要的生产实践意义。通过筛选水稻资源,发现了抗除草剂材料金粳818,其ALS基因编码区第1880位碱基存在一个由G到A的碱基变异,导致丝氨酸突变为天冬酰胺,从而具有除草剂抗性。本研究基于该位点的碱基变异,设计了11条等位基因特异PCR(allelic-specific PCR,AS-PCR)引物,经过优化筛选,获得两个引物组合F1N(S1/S9)和F1M(S1/S10),将该标记命名为AS-ALS。利用F2群体及其亲本和杂交种,结合AS-ALS标记检测和除草剂抗性分析,结果表明感除草剂ALS-G等位基因型只能被F1N引物对有效扩增,抗除草剂ALS-A等位基因型只能被F1M引物对有效扩增,而杂合基因型能同时被两对引物F1N和F1M扩增,ALS-A纯合或杂合等位基因型都表现抗除草剂,ALS-G纯合基因型表现感除草剂。因此本研究开发的标记能有效区分除草剂抗性基因的3种基因型,基因型与表型完全对应。该标记用于回交育种,可以选择ALS-A杂合基因型单株,剔除ALS-G纯合等位基因型,在自交的F2保留ALS-A纯合基因型单株,连续自交,能快速获得除草剂抗性稳定的水稻材料。该除草剂抗性基因的功能标记还可用于咪唑啉酮类除草剂抗性资源筛选。

水稻和稗草ALS活性测定及农美利选择性机理研究

乙酰乳酸合成酶 (ALS)是除草剂主要作用靶标 ,新型除草剂农美利 (bispyribac sodium)即是通过抑制ALS的活性使支链氨基酸的生物合成受阻而达到除草目的。作者利用ALS脱羧产物 3 羟基丁酮与肌酸及甲萘酚形成的复合物 ,采用比色法测定ALS活性和农美利的抑制作用 ,以明确农美利对水稻和稗草的选择性机理。研究结果表明 ,水稻和稗草体内ALS酶促反应复合产物的吸收峰为 52 0nm ;ALS活性在不同水稻品种和不同杂草体内各不相同 ,不同水稻品种依次为汕优 63、秀水 11>祥湖 84 >嘉育 2 93,不同杂草依次为稗草、鳢肠 >异型莎草 >千金子。体内法测定农美利对植物体内ALS的抑制程度结果显示 ,水稻ALS受抑制快于稗草 ,恢复也快 ;不同水稻品种ALS以嘉育 2 93受抑制较快 ,秀水 11抑制程度较大 ,而祥湖 84和汕优 63受抑制慢且程度低。体外法测定不同化合物对ALS抑制程度 。

磺酰脲类除草剂对抗、感性慈姑ALS活性的影响

2000—2002年,延边地区稻田发现了抗磺酰脲类除草剂苄嘧磺隆的生态型慈姑。与敏感型相比,苄嘧磺隆、吡嘧磺隆对抗药性慈姑乙酰乳酸合成酶活性的抑制效果很低,抗药性慈姑乙酰乳酸合成酶对苄嘧磺隆和吡嘧磺隆的抗性系数(RI50/SI50)值分别为57.3、20.0,并存在交互抗药性。确认抗药性生态型慈姑的抗药性是由其乙酰乳酸合成酶对苄嘧磺隆、吡嘧磺隆毒性反应钝化所致。

ALS抑制剂类除草剂抗性水稻功能标记的开发与验证

【目的】培育具有乙酰乳酸合酶(ALS)抑制剂类除草剂抗性的品种是防治水稻直播田杂草危害最经济、有效的途径之一,而利用分子标记辅助选择有助于提高其品种选择效率。【方法】在明确除草剂抗性突变体黄华占M-1 ALS基因编码区碱基差异的基础上,利用四引物扩增受阻突变体系PCR(Tetra-primer ARMS-PCR)技术,设计引物对不同品种(品系)和淮稻5号/黄华占M-1的F2群体进行基因型检测,并结合除草剂的田间试验对标记的准确性进行验证。【结果】ALS基因编码区序列比对分析表明,尽管籼、粳稻之间具有多处差异碱基,但只有第1642和1643碱基TG到AT的变异能导致位于高度保守域的第548位编码氨基酸由色氨酸突变为甲硫氨酸,进而使水稻产生对ALS抑制剂类除草剂的抗性。依据PCR扩增产物的电泳带型,可以准确区分出3种不同的基因型,其基因型与苗期除草剂抗性的表型完全一致。【结论】利用Tetra-primer ARMS-PCR技术,可以实现对两个连续变异碱基位点基因型的快速检测,从而加快ALS抑制剂类除草剂抗性水稻品种的选育进程。

油菜抗咪唑啉酮类除草剂基因BnALS1R等位基因特异PCR标记的开发与应用

油菜抗咪唑啉酮类除草剂基因BnALS1R是从抗性突变体M9中克隆获得,抗性基因BnALS1R与野生型基因BnALS1存在1处SNP,即乙酰乳酸合酶第638位丝氨酸残基被天冬酰胺酸替代。为获得油菜抗除草剂基因BnALS1R的分子标记,根据该处点突变,结合获得的BnALS3与BnALS1序列,开发30条等位基因特异PCR(allele-specific PCR,AS-PCR)引物,采用筛选出的3条AS-PCR引物在F2、BC1和BC2群体中进行PCR扩增。结果表明,该标记有效检测出群体中存在的3种基因型,其分离比分别为1∶2∶1、1∶1、1∶1,均遵循单基因遗传规律。应用该标记对获得的抗性恢复系进行PCR扩增,结果发现所有抗性恢复系均能扩增出抗性基因BnALS1R目的条带,表明3条标记引物可应用于抗性基因的检测。AS-PCR标记的获得将促进以抗性基因进行油菜抗除草剂分子标记辅助选择育种。

距离比较法(DISCO)构建ALS抑制剂药效团模型

在乙酰乳酸合成酶 (ALS)三维结构未知的情况下 ,利用距离比较法 (DISCO) ,将 10个结构特征具有代表性的ALS抑制剂的分子构象进行叠合 ,建立了可能的药效团模型 ,并初步验证了模型的可靠性 .

油菜乙酰乳酸合酶突变体S638N的酶学特性及其对ALS类除草剂的抗性

在对油菜抗咪唑啉酮类除草剂基因Bn ALS1R克隆与功能验证基础上,为比较抗性基因编码的乙酰乳酸合酶突变体S638N酶学特性及其对ALS类除草剂抗性与野生型的差异,构建基因原核表达载体,在大肠杆菌中表达S638N和野生型的重组融合蛋白。SDS-PAGE和Western blot分析表明,S638N和野生型均能表达出约74 k D的特异性重组蛋白。纯化目的蛋白,在不同温度和pH条件下,测定S638N和野生型的酶活性。结果显示,温度和pH对突变酶活性的影响与野生型相同,表现为先升后降,在37℃、pH 7.0条件下催化活性均最高。同时,该突变酶的酶学动力学参数Km和Vmax与野生型没有显著差异,其对3个辅助因子的响应曲线也与野生型类似,缺少其中任何一个辅助因子均使突变酶S638N基本都没有活性。然而,突变酶S638N对IMI类除草剂抗性显著高于野生型,而对Su类除草剂敏感性和野生型相同。因此,突变酶S638N具有对IMI类除草剂的专一抗性,但未改变酶学反应特征。

2-嘧啶氧基-N-芳基苄胺类化合物的ALS抑制活性的QSAR研究

乙酰乳酸合成酶(ALS)或乙酰羟酸合成酶(AHAS)存在于植物的生长过程中,很多此类酶的抑制剂实际上作为除草剂被广泛用于农业生产中.生物活性测试结果表明,2-嘧啶氧基-N-芳基苄胺类化合物对ALS具有一定的抑制活性.在此基础上,我们用两种三维定量构效关系(3D-QSAR)研究方法:比较分子立场分析(CoMFA)和比较分子相似性指数分析(CoMSIA),对该类化合物进行了3D-QSAR研究,并建立了相关的预测模型.其中,CoMFA模型的交叉验证相关系数(rcv2)为0.801,非交叉验证相关系数(r2)为0.947,标准偏差(s)为0.136,F值为133.371.CoMSIA模型的rcv2为0.744,r2为0.883,s为0.202,F值为56.472.计算结果表明,2-嘧啶氧基-N-芳基苄胺类化合物与ALS抑制活性有一定的相关性.获得的CoMFA和CoMSIA模型,将应用于指导该类化合物的设计.

甘蓝型油菜抗苯磺隆突变体ALS基因分析与SNP标记

为筛选新型甘蓝型油菜抗苯磺隆除草剂的等位基因特异分子标记,以EMS诱变的甘蓝型油菜中双9号M2群体(约30 000单株)为材料,通过苯磺隆喷药处理筛选到3株抗除草剂突变体(分别记为K1、K4、K5)。利用PCR技术克隆到抗性突变体相应的Bn ALS1、Bn ALS2、Bn ALS3基因,序列分析结果表明,K1和K4的Bn ALS3基因序列发生碱基突变,其中第+535位C碱基突变为T,导致其编码的Bn ALS3蛋白第197位氨基酸由Pro突变为Ser;而K5的Bn ALS1基因序列第+544位C突变为T,导致其编码的Bn ALS1蛋白发生了Pro197Ser的改变(均以拟南芥ALS氨基酸序列为准)。其中K5抗性位点Bn ALS1∶Pro197Ser的改变属于首次报道。基于突变的基因序列设计了8对等位基因特异性PCR引物组合,有6对能用于区分抗性突变体和野生型材料,其中可检测K5的Bn ALS1基因SNP位点的引物有2对,可检测K1、K4突变体Bn ALS3基因SNP位点的引物有4对。

模拟设计抗药突变型ALS抑制物结构的研究进展

乙酰乳酸合成酶(ALS)是植物体内合成支链氨基酸过程中的关键酶类。生产上对以ALS为单一靶标的磺酰脲类等5大类除草剂的连续广泛使用,使杂草抗药性突变体大规模产生。本文从计算机辅助药物设计(CADD)的角度出发,综述通过同源模建、结构稳定性分析和分子动力学模拟(MDS)等方法,推测替代突变导致ALS构象变化的抗性机理,并根据模拟抑制物与野生和突变型ALS结合物理参数的差异,如构象标准差、旋转半径、结合自由能,设计理论上对突变型ALS有抑制作用的化合物新结构,以期为抗药突变型靶标与其抑制剂之间的分子作用机理研究和反抗类除草剂的开发提供参考。

苯磺隆对甘蓝型油菜的杀雄效果及对其靶标ALS活性的影响

研究了叶面喷施杀雄剂苯磺隆对甘蓝型油菜品种宁油18号和096030的杀雄效果。结果表明:叶面喷施0.2μg/mL苯磺隆2次(每次8～10 mL/株)可诱导油菜100%的全不育株率,使整个花期不育,相对结实率仅约4%;经0.2μg/mL苯磺隆处理后,2种油菜中的乙酰乳酸合成酶(acetolactate synthase,ALS)的活性均较对照显著降低,ALS相对酶活分别为40.63%和37.23%。因此,油菜中ALS活性的变化有可能作为快速鉴定和预测杀雄剂苯磺隆的喷药量是否达到合理范围的指标。