抗苯磺隆播娘蒿抗性机理及抗性突变对乙酰乳酸合成酶功能影响
发布时间：2019-06-25 00:12
【摘要】：播娘蒿(Descurainia sophia L.)属于十字花科一年或越年生杂草,是为害我国麦田的恶性杂草之一。苯磺隆是磺酰脲类除草剂,主要用于防治麦田播娘蒿等阔叶杂草,从1988年在我国登记使用,经过连续多年的使用,播娘蒿已对苯磺隆产生了严重抗药性。本研究以采自北京、河南、河北和山东省份的麦田播娘蒿为研究试材,测定了不同播娘蒿种群对苯磺隆的抗性水平和抗性机理,将具有不同乙酰乳酸合成酶(ALS)突变型播娘蒿种群繁殖、纯化为具有单一突变类型的种群,并以此为研究对象进一步研究了不同ALS突变型对ALS除草剂交互抗性、ALS酶功能和支链氨基酸含量的影响,探讨了利用分子生物学技术鉴定抗性植株的快速检测方法,主要研究结果如下:1.抗苯磺隆播娘蒿种群初步鉴定利用区分剂量法鉴定了 73个播娘蒿种群对苯磺隆的抗性,结果表明:28个为抗性种群,18个种群具有潜在的抗性风险,27个为敏感种群。2.抗性播娘蒿ALS基因突变多样性研究播娘蒿ALS基因突变多样性鉴定结果表明,播娘蒿体内存在两个ALS基因,分别为ALS1和 ALS2,ALS1 基因上 Pro197 突变成 Leu、Ser、Thr、His 或 Tyr,Asp376 突变成 Glu,Trp574突变成Leu,ALS2基因上Pro197突变成Ser或Thr。在28个抗性突变种群中Pro-197-Leu突变比例为 39.3%,Pro-197-Ser 突变比例为 28.6%,Pro-197-Thr 突变比例为 28.6%,Pro-197-His 突变比例为 10.7%,Pro-197-Tyr 突变比例为 3.6%,Asp-376-Glu 突变比例为 3.6%,Trp-574-Leu 突变比例为7.1%,其中Pro-197-Tyr突变在播娘蒿上是首次鉴定并发现。Trp-574-Leu(in ALS1)突变和Pro-197-Thr(in ALS2)突变发生在同一抗性植株中,这是首次发现的在田间自然选择条件下而形成的双突变杂草。3.不同ALS突变型对播娘蒿抗性和交互抗性影响研究为了减少不同种群间遗传背景差异,对具有不同ALS突变型的播娘蒿进行单株繁殖,获得了具有不同突变纯合子的播娘蒿种群,分别为pHB08(Pro-197-Leu)、pHB22(Pro-197-Ser)、pHB23(Pro-197-Thr)、pHB24(Pro-197-His)、pHB25(Asp-376-Glu)和 pHB42(Trp-574-Leu)种群,以及 Trp-574-Leu(in ALS1)突变和 Pro-197-Thr(in ALS2)突变的 pHB30 种群。盆栽法测定了纯化种群 pHB08、pHB22、pHB23、pHB24、pHB25、pHB42 和 pHB30 对苯磺隆的抗性水平,结果表明纯化的播娘蒿对苯磺隆产生了高水平的抗性,其中pHB25(Asp-376-Glu)和 pHB30(Trp-574-Leu + Pro-197-Thr)种群比 pHB08(Pro-197-Leu)、pHB22(Pro-197-Ser)、pHB23(Pro-197-Thr)、pHB24(Pro-197-His)和 pHB42(Trp-574-Leu)种群对苯磺隆的抗性水平更高(789.3-815.0vs 152.3-366.3)。不同ALS突变型播娘蒿对ALS除草剂的交互抗性测定结果表明,Pro-197-Leu、Pro-197-Ser、Pro-197-Thr和Pro-197-His突变的播娘蒿种群对氯吡嘧磺隆(SU)、唑嘧磺草胺(TP)和氟唑磺隆(SCT)产生了交互抗性,对咪唑乙烟酸(IMI)和嘧啶肟草醚(PTB)没有交互抗性;而Asp-376-Glu突变的pHB25种群,Trp-574-Leu突变的pHB42种群和双突变的pHB30种群对氯吡嘧磺隆、唑嘧磺草胺、氟唑磺隆、咪唑乙烟酸和嘧啶肟草醚五类ALS除草剂都产生了交互抗性。4.不同ALS突变型对播娘蒿ALS功能影响研究乙酰乳酸合成酶催化活性和对苯磺隆敏感性测定结果表明,和敏感种群相比,抗性种群pHB08、pHB22、pHB23、pHB24、pHB25和pHB42乙酰乳酸合成酶对苯磺隆的敏感性分别下降了 250.0、205.8、263.7、708.4、2844.7 和 485.3 倍,且 pHB08、pHB22、pHB23 和 pHB24 种群ALS催化活性有显著性降低(11%-21%),pHB25种群ALS催化活性有显著性升高(26%),而pHB42种群ALS催化活性没有显著性差异。酶动力学研究结果表明,和敏感种群相比,抗性种群pHB08、pHB22、pHB23、pHB24、pHB25和pHB42乙酰乳酸合成酶对底物的Km值和最大反应速率Vmax值都有显著性下降,Km值下降了0.63-0.87 倍,Vmax值下降了 0.30-0.91 倍。反馈抑制研究表明,Pro-197-Leu、Pro-197-Ser、Pro-197-Thr和Pro-197-His突变种群乙酰乳酸合成酶对亮氨酸的反馈抑制减弱,对缬氨酸和异亮氨酸的反馈抑制则没有影响,Asp-376-Glu突变种群和Trp-574-Leu突变种群乙酰乳酸合成酶对亮氨酸、缬氨酸和异亮氨酸的反馈抑制都减弱。5.不同ALS突变型对播娘蒿体内支链氨基酸含量影响不同ALS突变型播娘蒿体内支链氨基酸含量测定结果表明,Pro-197-Leu、Pro-197-His、Pro-197-Ser和Pro-197-Thr突变抗性植株体内亮氨酸和异亮氨酸含量和敏感植株没有差异(Pro-197-Ser突变植株体内异亮氨酸含量比敏感植株较低),Asp-376-Glu突变和Trp-574-Leu突变抗性植株体内亮氨酸和缬氨酸含量比敏感植株显著性增加。6.抗苯磺隆播娘蒿快速检测方法研究针对敏感与抗性播娘蒿植株在ALS基因Pro197、Asp376或Trp574位点的碱基差异,用CAPS方法设计BsmFI、MboI和BsrDI酶切检测了 48株播娘蒿植株的靶标抗性,结果表明,此方法准确率为100%,可用于快速检测靶标突变的抗性播娘蒿植株。
[Abstract]:Artemisia sophiia L. is one of the most malignant weeds in the wheat field of China. Benuron is a kind of herbicide, which is mainly used for preventing and controlling broad-leaf weeds such as artemisia argyi and other broad-leaf weeds in the wheat field, and has been used in our country in 1988. In this study, a study was conducted to study the resistance level and the resistance mechanism of Artemisia annua population to benuron from Beijing, Henan, Hebei and Shandong provinces. The effects of different ALS mutants on the interaction resistance of ALS, the function of ALS and the content of branched-chain amino acids were further studied by using the method of molecular biology to identify the resistant plants. The main results are as follows:1. The results of the preliminary identification of the population of the artemisia-bentauroides were identified by means of a different dose method. The results showed that 28 were resistant to the population,18 of them had potential risk of resistance, and 27 were sensitive. The results showed that there are two ALS genes, respectively, ALS1 and ALS2, and Pro197 in the ALS1 gene is mutated to Leu, Ser, Thr, His or Tyr, and Asp376 is mutated to Glu, and Trp574 is mutated to Leu, The mutation of Pro-197-Leu in 28 resistant mutant populations was 39.3%, the ratio of Pro-197-Ser was 28.6%, the ratio of Pro-197-Thr was 28.6%, the ratio of Pro-197-His was 10.7%, the ratio of Pro-197-Tyr was 3.6%, the mutation ratio of Asp-376-Glu was 3.6%, and the ratio of Trp-574-Leu was 7.1%. The Pro-197-Tyr mutation was first identified and found on the artemisia. The mutation of Trp-574-Leu (in ALS1) and the mutation of Pro-197-Thr (in ALS2) occurred in the same resistance plant, which was the first discovered double-mutant weed under field natural selection conditions. In order to reduce the difference of genetic background among different populations, the effects of different ALS mutants on the resistance and the interaction resistance of the bung-bridesmaids were studied. In order to reduce the genetic background difference among different populations, a single-plant propagation was carried out on the mugwort with different mutation homozygotes, which were pHB08 (Pro-197-Leu), pHB22 (Pro-197-Ser), pHB23 (Pro-197-Thr), pHB24 (Pro-197-His), respectively. PHB25 (Asp-376-Glu) and pHB42 (Trp-574-Leu) populations, as well as the pHB30 population of the Trp-574-Leu (in ALS1) mutation and the Pro-197-Thr (in ALS2) mutation. The resistance of pHB08, pHB22, pHB23, pHB24, pHB25, pHB42, and pHB30 to benuron was determined by pot-pot method. The results showed that the purified artemisia annua had a high level of resistance to benuron, among which pHB25 (Asp-376-Glu) and pHB30 (Trp-574-Leu + Pro-197-Thr) were higher than pHB08 (Pro-197-Leu), pHB22 (Pro-197-Ser), pHB23 (Pro-197-Thr). The resistance of pHB24 (Pro-197-His) and pHB42 (Trp-574-Leu) to benuron was higher (789.3-815.0 vs. 152.3-366.3). The results of the interactive resistance of the different ALS mutants to the ALS of the ALS showed that the populations of the wild Artemisia annua (L.), Pro-197-Leu, Pro-197-Ser, Pro-197-Thr and Pro-197-His were resistant to the interaction of chlormetsulfuron (SU), metasolachlor (TP), and flumetsulfuron (SCT). the pHB25 population of the Asp-376-Glu mutation, the pHB42 population of the Trp-574-Leu mutation, Medemiconicotinic acid and the five-class ALS herbicides of the trimedoxime are resistant to the interaction. The effect of the different ALS mutant on the function of the sophiolite ALS was studied. The results showed that the resistant population was pHB08, pHB22, pHB23, pHB24 compared with the sensitive population. The sensitivity of pHB25 and pHB42 B-lactic acid synthase to benuron decreased by 250.0, 205.8, 263.7, 708.4, 2844.7 and 485.3 times, respectively, and the catalytic activity of pHB08, pHB22, pHB23 and pHB24 was significantly lower (11%-21%), and the catalytic activity of pHB25 population was significantly higher (26%), while the catalytic activity of pHB42 population was not significantly different. The results showed that the Km and the maximum reaction rate of pHB08, pHB22, pHB23, pHB24, pHB25, pHB42 and pHB42, pHB24, pHB25 and pHB42 were significantly lower than those in the sensitive population, and the Km value decreased by 0.63-0.87-fold and the Vmax value decreased by 0.30-0.91-fold. The feedback inhibition study indicated that Pro-197-Leu, Pro-197-Ser, Pro-197-Thr and Pro-197-His mutant population B-lactic acid synthase reduced the feedback inhibition of leucine, and the feedback inhibition on the leucine and isoleucine was not affected, and the Asp-376-Glu mutant population and the Trp-574-Leu mutant population B-lactic acid synthetase pair leucine, The inhibition of the feedback of theanine and isoleucine is reduced. The results of the determination of the content of the branched amino acids in the mugwort of different ALS in different ALS mutants showed that Pro-197-Leu, Pro-197-His, In the Pro-197-Ser and Pro-197-Thr mutant, there was no difference in the content of leucine and isoleucine in the susceptible plants (the content of isoleucine in the Pro-197-Ser mutant plants was lower than that of the sensitive plants), and the levels of leucine and theanine in the mutant resistant plants of the Asp-376-Glu and Trp-574-Leu were significantly higher than that of the sensitive plants. In this paper, the target resistance of 48 strains of Artemisia argyi plant was detected by means of the CAPS method. The results showed that the accuracy of the method was 100%. And can be used for rapidly detecting the resistance of the target mutation.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S451.2
本文编号：2505484
[Abstract]:Artemisia sophiia L. is one of the most malignant weeds in the wheat field of China. Benuron is a kind of herbicide, which is mainly used for preventing and controlling broad-leaf weeds such as artemisia argyi and other broad-leaf weeds in the wheat field, and has been used in our country in 1988. In this study, a study was conducted to study the resistance level and the resistance mechanism of Artemisia annua population to benuron from Beijing, Henan, Hebei and Shandong provinces. The effects of different ALS mutants on the interaction resistance of ALS, the function of ALS and the content of branched-chain amino acids were further studied by using the method of molecular biology to identify the resistant plants. The main results are as follows:1. The results of the preliminary identification of the population of the artemisia-bentauroides were identified by means of a different dose method. The results showed that 28 were resistant to the population,18 of them had potential risk of resistance, and 27 were sensitive. The results showed that there are two ALS genes, respectively, ALS1 and ALS2, and Pro197 in the ALS1 gene is mutated to Leu, Ser, Thr, His or Tyr, and Asp376 is mutated to Glu, and Trp574 is mutated to Leu, The mutation of Pro-197-Leu in 28 resistant mutant populations was 39.3%, the ratio of Pro-197-Ser was 28.6%, the ratio of Pro-197-Thr was 28.6%, the ratio of Pro-197-His was 10.7%, the ratio of Pro-197-Tyr was 3.6%, the mutation ratio of Asp-376-Glu was 3.6%, and the ratio of Trp-574-Leu was 7.1%. The Pro-197-Tyr mutation was first identified and found on the artemisia. The mutation of Trp-574-Leu (in ALS1) and the mutation of Pro-197-Thr (in ALS2) occurred in the same resistance plant, which was the first discovered double-mutant weed under field natural selection conditions. In order to reduce the difference of genetic background among different populations, the effects of different ALS mutants on the resistance and the interaction resistance of the bung-bridesmaids were studied. In order to reduce the genetic background difference among different populations, a single-plant propagation was carried out on the mugwort with different mutation homozygotes, which were pHB08 (Pro-197-Leu), pHB22 (Pro-197-Ser), pHB23 (Pro-197-Thr), pHB24 (Pro-197-His), respectively. PHB25 (Asp-376-Glu) and pHB42 (Trp-574-Leu) populations, as well as the pHB30 population of the Trp-574-Leu (in ALS1) mutation and the Pro-197-Thr (in ALS2) mutation. The resistance of pHB08, pHB22, pHB23, pHB24, pHB25, pHB42, and pHB30 to benuron was determined by pot-pot method. The results showed that the purified artemisia annua had a high level of resistance to benuron, among which pHB25 (Asp-376-Glu) and pHB30 (Trp-574-Leu + Pro-197-Thr) were higher than pHB08 (Pro-197-Leu), pHB22 (Pro-197-Ser), pHB23 (Pro-197-Thr). The resistance of pHB24 (Pro-197-His) and pHB42 (Trp-574-Leu) to benuron was higher (789.3-815.0 vs. 152.3-366.3). The results of the interactive resistance of the different ALS mutants to the ALS of the ALS showed that the populations of the wild Artemisia annua (L.), Pro-197-Leu, Pro-197-Ser, Pro-197-Thr and Pro-197-His were resistant to the interaction of chlormetsulfuron (SU), metasolachlor (TP), and flumetsulfuron (SCT). the pHB25 population of the Asp-376-Glu mutation, the pHB42 population of the Trp-574-Leu mutation, Medemiconicotinic acid and the five-class ALS herbicides of the trimedoxime are resistant to the interaction. The effect of the different ALS mutant on the function of the sophiolite ALS was studied. The results showed that the resistant population was pHB08, pHB22, pHB23, pHB24 compared with the sensitive population. The sensitivity of pHB25 and pHB42 B-lactic acid synthase to benuron decreased by 250.0, 205.8, 263.7, 708.4, 2844.7 and 485.3 times, respectively, and the catalytic activity of pHB08, pHB22, pHB23 and pHB24 was significantly lower (11%-21%), and the catalytic activity of pHB25 population was significantly higher (26%), while the catalytic activity of pHB42 population was not significantly different. The results showed that the Km and the maximum reaction rate of pHB08, pHB22, pHB23, pHB24, pHB25, pHB42 and pHB42, pHB24, pHB25 and pHB42 were significantly lower than those in the sensitive population, and the Km value decreased by 0.63-0.87-fold and the Vmax value decreased by 0.30-0.91-fold. The feedback inhibition study indicated that Pro-197-Leu, Pro-197-Ser, Pro-197-Thr and Pro-197-His mutant population B-lactic acid synthase reduced the feedback inhibition of leucine, and the feedback inhibition on the leucine and isoleucine was not affected, and the Asp-376-Glu mutant population and the Trp-574-Leu mutant population B-lactic acid synthetase pair leucine, The inhibition of the feedback of theanine and isoleucine is reduced. The results of the determination of the content of the branched amino acids in the mugwort of different ALS in different ALS mutants showed that Pro-197-Leu, Pro-197-His, In the Pro-197-Ser and Pro-197-Thr mutant, there was no difference in the content of leucine and isoleucine in the susceptible plants (the content of isoleucine in the Pro-197-Ser mutant plants was lower than that of the sensitive plants), and the levels of leucine and theanine in the mutant resistant plants of the Asp-376-Glu and Trp-574-Leu were significantly higher than that of the sensitive plants. In this paper, the target resistance of 48 strains of Artemisia argyi plant was detected by means of the CAPS method. The results showed that the accuracy of the method was 100%. And can be used for rapidly detecting the resistance of the target mutation.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S451.2
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