黑龙江省稻田野慈姑对丙嗪嘧磺隆抗性相关研究

通过整株盆栽法研究黑龙江省佳木斯市汤原县(种群R1)、856农场(种群R2)、密山市(种群R3)3个水稻田野慈姑种群对丙嗪嘧磺隆的抗性水平,采用分子生物学技术分析3个野慈姑种群在靶标酶基因上的差异,确定3个野慈姑种群对丙嗪嘧磺隆和苄嘧磺隆是否存在交互抗性。结果显示,黑龙江R1、R2、R3种群抗性指数(RI)分别为11.92、22.68、35.99。与敏感的七台河种群S相比,R1、R2、R3的ALS基因均在Pro_(197)位发生不同突变。R1种群为Thr_(197)取代了Pro_(197);R2、R3种群为Ser_(197)取代了Pro_(197),ALS基因的突变是其产生抗性的主要原因。3个野慈姑种群对丙嗪嘧磺隆和苄嘧磺隆存在交互抗性。     

辽宁省稻田野慈姑对苄嘧磺隆的抗药性

为明确辽宁省不同稻田区野慈姑对苄嘧磺隆的抗性水平,整株测定了辽宁省大石桥(种群R1)、海城(种群R2)、苏家屯(种群R3)和开原(种群R4)共4个水稻产区野慈姑对苄嘧磺隆的抗性水平,并离体测定了各种群叶片体内乙酰乳酸合成酶(ALS)对苄嘧磺隆的敏感性。结果显示,种群R1和R2的抗药性相对较高,抗性指数分别为76.99和49.94,种群R3和R4抗性相对较低,抗性指数分别为12.48和16.91;离体测定结果表明较高水平的ALS活性可能与是否产生抗药性无关,种群R1、R2、R3、R4的抗性指数分别为81.86、67.48、10.56、24.86;抗药性程度依次为R1R2R4R3。表明4个水稻产区野慈姑对苄嘧磺隆均产生了抗药性,而其体内ALS活性降低可能是产生抗药性的原因之一。     

野慈姑对吡嘧磺隆抗性的分子机理

野慈姑是我国稻田危害最严重的杂草之一。以黑龙江稻田采集的野慈姑(Sagittaria trifolia Linn.)为研究对象,通过室内生物测定和分子克隆技术,对野慈姑的吡嘧磺隆抗性水平进行检测,并从靶标位点突变的角度解释抗性产生原因。结果表明,采自哈尔滨稻田的2个野慈姑种群N03及N06均为高抗种群,抗性达到4倍剂量以上。经比对2个抗性种群的靶标酶ALS基因发现,Pro197的脯氨酸分别被亮氨酸、丝氨酸取代,该位点的突变可能是野慈姑种群对磺酰脲类除草剂产生抗药性的主要原因。     

抗苄嘧磺隆雨久花乙酰乳酸合成酶突变的研究

本研究采用cDNA末端快速扩增技术(RACE)结合RT-PCR方法克隆抗苄嘧磺隆雨久花生物型和敏感性雨久花生物型乙酰乳酸合成酶(ALS)基因cDNA序列,并对测序结果进行比对分析。结果表明:与敏感性的雨久花ALS相比,公主岭(GZL)抗性生物型中第197位脯氨酸突变为组氨酸,第556位亮氨酸突变为苯丙氨酸;柳河(LH)抗性生物型中第358位天冬酰胺突变为天冬氨酸;磐石市(PS)抗性生物型中第525位缬氨酸突变为异亮氨酸。分析表明,高度保守区Domain A的第197位氨基酸残基的突变可能是导致公主岭稻区雨久花产生抗药性的主要原因之一,而其他抗性生物型抗性产生的原因有待进一步研究。     

多花水苋对苄嘧磺隆的抗性水平及靶标抗性机制

为明确水稻田杂草多花水苋Ammannia multiflora对乙酰乳酸合成酶 (ALS) 抑制剂类除草剂苄嘧磺隆的抗性水平和抗性分子机制,采用整株水平测定法,测定了采自江苏省扬州市田间的多花水苋疑似抗性种群 (YZ-R) 对苄嘧磺隆的抗性指数,并分析了YZ-R种群和相对敏感种群 (YZ-S)多花水苋ALS酶对苄嘧磺隆的敏感性差异,同时比较了YZ-R和YZ-S种群ALS基因的核苷酸序列差异。结果表明:YZ-R种群多花水苋对苄嘧磺隆已表现出高水平抗性,其抗性指数 (RI) 为40.6;苄嘧磺隆对YZ-R种群ALS酶活性的抑制中浓度 (I₅₀) 为0.087 μmol/L,对YZ-S种群的I₅₀值为0.0028 μmol/L,其抗性指数为31.1。通过PCR扩增获得了多花水苋ALS基因的部分序列,该序列包含了已报道的8个氨基酸突变位点。ALS基因序列比对分析发现,YZ-R种群多花水苋植株ALS基因第197 位氨基酸由脯氨酸 (CCT) 突变为丝氨酸 (TCT)。研究表明,ALS基因发生脯氨酸 (Pro)-197-丝氨酸 (Ser) 的突变,导致多花水苋ALS酶对苄嘧磺隆的敏感性下降,是多花水苋YZ-R种群对苄嘧磺隆产生高水平抗性的主要原因。     

抗精噁唑禾草灵和甲基二磺隆看麦娘ACCase和ALS基因突变

为明确看麦娘Alopecurus aequalis抗性种群YL的靶标抗性机制,采用基因克隆法对看麦娘抗性和敏感种群间乙酰辅酶A羧化酶(ACCase)和乙酰乳酸合成酶(ALS)基因序列进行扩增、克隆和测序,比对二者ACCase和ALS基因序列的差异,探寻其产生抗药性突变的基因位点,同时测定该突变型抗性种群YL对不同ACCase和ALS抑制剂类除草剂的交互抗性。结果显示,与看麦娘敏感种群TL相比,抗性种群YL的ACCase基因CT区域第2 041位氨基酸由异亮氨酸(ATT)突变为天冬酰胺酸(AAT),ALS基因Domain A区域第197位氨基酸由脯氨酸(CCC)突变为精氨酸(CGC)。看麦娘抗性种群YL对ACCase抑制剂炔草酯产生了高水平抗性,抗性倍数为43.96,对高效氟吡甲禾灵和精喹禾灵产生了中等水平抗性,抗性倍数分别为18.33和15.87,对唑啉草酯、烯草酮和烯禾啶较敏感;对ALS抑制剂氟唑磺隆产生了低水平抗性,抗性倍数为8.39,对啶磺草胺和咪唑乙烟酸较敏感。表明ACCase基因第2 041位和ALS基因第197位氨基酸突变是导致看麦娘抗性种群YL对精噁唑禾草灵和甲基二磺隆同时产生抗性的重要原因之一。     

东北稻田野慈姑对苄嘧磺隆抗药性研究

为了明确东北不同稻田野慈姑(Sagittaria trifolia L.)对除草剂苄嘧磺隆的抗性水平,通过整株剂量反应和离体靶标酶活性差异测定了辽宁省、吉林省和黑龙江省11个野慈姑种群的抗性水平。结果表明,供试11个种群整株剂量测定结果均对苄嘧磺隆产生了高水平抗性,且2015—2016年供试种群的抗性水平未产生显著变化。其中,沈阳种群的抗性水平最高,2015年抗性指数为155.96,2016年抗性指数达161.54;离体ALS酶活性测定结果显示,舒兰种群和哈尔滨种群抗性指数(2015年分别为1.67和1.10,2016年分别为1.66和1.12)与敏感种群无显著差异,其他9个种群其抗性趋势与整株剂量反应结果相一致。数据还表明,ALS酶活性降低可能是使沈阳种群、榆树种群、绥化种群等9个野慈姑抗性种群产生抗药性的主要原因。     

耳叶水苋对乙酰乳酸合成酶抑制剂的抗性及其分子机制初探

近年来长江下游地区稻田耳叶水苋Ammannia arenaria H.B.K.危害十分严重。采用盆栽法首次测定了耳叶水苋对苄嘧磺隆等药剂的抗性水平,同时分析了其抗性和敏感种群间乙酰乳酸合成酶(ALS)基因的DNA序列及其RNA表达差异。结果表明:采自浙江嘉兴(JX110)、江苏苏州(JS039)、浙江宁波(NB0143-05)和安徽广德(AH014)的耳叶水苋生物型对苄嘧磺隆的抗性指数(RI)分别为67.90、17.59、44.63和8.37,对苄嘧磺隆表现出中高水平抗性的生物型对五氟磺草胺、双草醚及咪唑乙烟酸也产生了低水平的抗性。获得了耳叶水苋ALS基因全长核苷酸序列2235 bp,编码667个氨基酸,仅发现NB0143-05等3种抗性生物型ALS酶的氨基酸序列非保守区第93位的亮氨酸被脯氨酸取代。然而,NB0143-05的ALS酶对ALS抑制剂的敏感性大幅度降低(RI 37.04),且在苄嘧磺隆处理后4 d的ALS基因表达量是敏感生物型(HZ001)的1.86倍。这表明,ALS酶对药剂的敏感性降低以及被苄嘧磺隆诱导后ALS基因表达量显著增加,很可能是耳叶水苋生物型NB0143-05对ALS抑制剂产生抗性的原因。     

河南省部分地区麦田荠菜对苯磺隆的抗性水平及抗性靶标分子机制

为明确河南省部分地区麦田荠菜Capsella bursa-pastoris对苯磺隆的抗性水平及抗性靶标分子机制,采用整株生物法测定了12个荠菜种群的抗性水平,并对乙酰乳酸合成酶(acetolactate synthase,ALS)离体活性和ALS基因突变进行了测定分析。结果表明,商丘市民权县花园村(MQ)、周口市西华县小于楼村(XH)、平顶山市叶县穆寨村(YX)、许昌市长葛市董庄村(CG)采集的荠菜种群对苯磺隆产生了较高的抗性,GR_(50)分别为129.14、110.67、62.91和85.29 g/hm~2,抗性倍数分别为215.23、184.45、104.85和142.15倍;ALS离体活性测定所得I_(50)分别为5.85、4.87、1.38和3.83μmol/L,抗性倍数分别为83.57、69.57、19.71和54.71倍;其余8个种群的GR_(50)在0.60~2.86 g/hm~2之间,抗性倍数在1.00~4.77之间;I_(50)在0.07~0.37μmol/L之间,抗性倍数在1.00~5.29之间。荠菜种群MQ、XH的ALS基因Domain A区域第197位脯氨酸(CCT)均突变为丝氨酸(TCT),荠菜种群CG的第197位脯氨酸(CCT)突变为亮氨酸(CTT),表明靶标ALS基因突变是荠菜对苯磺隆产生抗性的重要原因之一,但荠菜种群YX的ALS基因保守区内暂未发现突变位点,其抗药性可能由其它原因造成。     

反枝苋对咪唑乙烟酸抗性水平及分子机制

为初步明确大豆田反枝苋对咪唑乙烟酸的抗药性水平,并从分子角度对抗药性机制进行解释,以我国四川成都和黑龙江嫩江采集的反枝苋种子为材料,通过琼脂法检测了反枝苋对咪唑乙烟酸的抗药性水平,并分别对R(嫩江抗性种群)和S(成都敏感种群)的乙酰乳酸合成酶(ALS)部分序列进行扩增和测序。皿内生测结果表明,成都种群的GI50为11.20,嫩江种群的GI50为52.26,其抗药性指数RI为4.67。分子检测结果表明,与S种群相比,R种群反枝苋ALS位于高度保守区Domain B编码574位氨基酸的基因发生突变,TGG突变为TTG,导致色氨酸被亮氨酸取代。ALS保守区域氨基酸的替换可能是嫩江反枝苋种群对咪唑乙烟酸产生抗性的重要原因之一。     

Variation in the gene encoding acetolactate-synthase in Lolium species and proactive detection of mutant, herbicide-resistant alleles

Lolium species (ryegrasses) are genetically highly variable plants that are both forage crops and major weeds across the globe. As weeds, they rapidly evolve resistance under the selective pressure of acetolactate-synthase (ALS) inhibitors, the most resistance-prone herbicide group. Quick and accurate diagnosis is therefore of importance to prevent resistance spread in ryegrass. To develop proactive molecular tools for the detection of mutant, resistant ALS alleles, we assessed variation in the ryegrass ALS gene. Sequencing the full 1929-bp ALS coding sequence in 59 plants from six distant locations revealed a total of 208 polymorphic nucleotide positions (one every 9.3 nucleotides). The heterogeneous distribution of synonymous and non-synonymous substitutions along the ALS coding sequence suggested that nucleotide variation of ALS is shaped by purifying and background selection. Using regions of the ALS coding sequence with a low number of polymorphic nucleotide sites, five derived cleaved amplified polymorphic sequence (dCAPS) assays were developed targeting codons crucial for herbicide sensitivity. These enabled the first detection in ryegrass of a Pro-197-Thr substitution that confers herbicide resistance. Most assays could also be used to genotype Festuca and Vulpia plants. These dCAPS assays should prove powerful tools for both resistance diagnosis and population genetics studies.     

小麦田看麦娘对甲基二磺隆的抗性水平及分子机制初探

看麦娘是中国长江中下游地区稻茬麦田的主要恶性杂草之一,甲基二磺隆是防治小麦田看麦娘等禾本科杂草的重要除草剂.该研究团队前期在安徽省凤台县小麦田采集到疑似抗性种群看麦娘(AHFT-01),为明确其对甲基二磺隆的抗性发生情况及潜在的抗性机制,采用温室盆栽法在整株水平上测定了该种群对甲基二磺隆及其他乙酰乳酸合成酶(ALS)抑...     

Mutations of the ALS gene endowing resistance to ALS-inhibiting herbicides in Lolium rigidum populations

BACKGROUND: In the important grass weed Lolium rigidum (Gaud.), resistance to ALS‐inhibiting herbicides has evolved widely in Australia. The authors have previously characterised the biochemical basis of ALS herbicide resistance in a number of L. rigidum biotypes and established that resistance can be due to a resistant ALS and/or enhanced herbicide metabolism. The purpose of this study was to identify specific resistance‐endowing ALS gene mutation(s) in four resistant populations and to develop PCR‐based molecular markers. RESULTS: Six resistance‐conferring ALS mutations were identified: Pro‐197‐Ala, Pro‐197‐Arg, Pro‐197‐Gln, Pro‐197‐Leu, Pro‐197‐Ser and Trp‐574‐Leu. All six mutations were found in one population (WLR1). Each Pro‐197 mutation conferred resistance to the sulfonylurea (SU) herbicide sulfometuron, whereas the Trp‐574‐Leu mutation conferred resistance to both sulfometuron and the imidazolinone (IMS) herbicide imazapyr. A derived cleaved amplified polymorphic sequences (dCAPS) marker was developed for detecting resistance mutations at Pro‐197. Furthermore, cleaved amplified polymorphic sequences (CAPS) markers were developed for detecting each of the six mutant resistant alleles. Using these markers, the authors revealed diverse ALS‐resistant alleles and genotypes in these populations and related them directly to phenotypic resistance to ALS‐inhibiting herbicides. CONCLUSION: This study established the existence of a diversity of ALS gene mutations endowing resistance in L. rigidum populations: 1–6 different mutations were found within single populations. At field herbicide rates, resistance profiles were determined more by the specific mutation than by whether plants were homo‐ or heterozygous for the mutation. Copyright © 2008 Society of Chemical Industry     

河南省多花黑麦草对ACCase和ALS抑制剂的抗性及其靶标基因突变分析

为明确河南省部分地区的多花黑麦草Lolium multiflorum种群对乙酰辅酶A羧化酶（acetylCoA carboxylase,ACCase）和乙酰乳酸合成酶（acetolactate synthase,ALS）抑制剂类除草剂的抗性水平和抗性机理,采用整株生物测定法测定采自新乡市和驻马店市的多花黑麦草种群对ACCase抑制剂类除草剂精噁唑禾草灵、炔草酯、唑啉草酯和ALS抑制剂类除草剂甲基二磺隆、氟唑磺隆、啶磺草胺的抗性水平,并对多花黑麦草ACCase和ALS靶标酶编码基因进行克隆及氨基酸序列比对,分析其靶标抗性机理。结果显示,与多花黑麦草敏感种群HNXX01相比,HNZMD04和HNXX05种群对6种除草剂均产生了抗性,HNZMD04种群对精噁唑禾草灵和啶磺草胺的相对抗性倍数分别为44.65和40.31,对炔草酯和氟唑磺隆的相对抗性倍数分别为11.91和11.93;HNXX05种群对精噁唑禾草灵和氟唑磺隆的相对抗性倍数分别为27.70和25.67。HNZMD04和HNXX05抗性种群的ACCase基因均发生了D2078G突变,2个种群的突变率分别为55%和70%;HNZMD04...     

Target-site basis for resistance to acetolactate synthase inhibitor in Water chickweed (Myosoton aquaticum L.)

Water chickweed is a widespread and competitive winter annual or biennial weed of wheat in China. One Water chickweed population (HN02) resistant to several acetolactate synthase (ALS) inhibitors was found in Henan province of China. Whole-plant bioassays showed that HN02 was high resistance to tribenuron (292.05-flod). In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to tribenuron. The I₅₀ value for HN02 was 85.53 times greater respectively than that of susceptible population (SD05). This altered ALS sensitivity in the resistant population was due to a mutation in the ALS gene resulting in a Pro197 to Ser substitution. Cross-resistance experiments indicated that HN02 exhibited various resistance patterns to pyrithiobac-sodium, florasulam and pyroxsulam, without resistance to imazethapyr. This is the first report of tribenuron-resistant Water chickweed in Henan province of China, target-site based resistance was established as being due to an insensitive form of ALS, resulting from a Pro to Ser substitution at amino acid position 197 in the ALS gene.     

Molecular characterisation of resistance to ALS-inhibiting herbicides in Hordeum leporinum biotypes

BACKGROUND: The acetolactate synthase (ALS)-inhibiting herbicide sulfosulfuron is registered in Australia for the selective control of Hordeum leporinum Link. in wheat crops. This herbicide failed to control H. leporinum on two farms in Western Australia on its first use. This study aimed to determine the level of resistance of three H. leporinum biotypes, identify the biochemical and molecular basis and develop molecular markers for diagnostic analysis of the resistance. RESULTS: Dose-response studies revealed very high level (>340-fold) resistance to the sulfonylurea herbicides sulfosulfuron and sulfometuron. In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to herbicide inhibition. This altered ALS sensitivity in the resistant biotypes was found to be due to a mutation in the ALS gene resulting in amino acid proline to serine substitution at position 197. In addition, two- to threefold higher ALS activities were consistently found in the resistant biotypes, compared with the known susceptible biotype. Two cleaved amplified polymorphic sequence (CAPS) markers were developed for diagnostic testing of the resistant populations. CONCLUSION: This study established the first documented case of evolved ALS inhibitor resistance in H. leporinum and revealed that the molecular basis of resistance is due to a Pro to Ser mutation in the ALS gene.