抗苯磺隆播娘蒿抗性機(jī)理及抗性突變對(duì)乙酰乳酸合成酶功能影響
發(fā)布時(shí)間：2019-06-25 00:12
【摘要】：播娘蒿(Descurainia sophia L.)屬于十字花科一年或越年生雜草,是為害我國(guó)麥田的惡性雜草之一。苯磺隆是磺酰脲類除草劑,主要用于防治麥田播娘蒿等闊葉雜草,從1988年在我國(guó)登記使用,經(jīng)過(guò)連續(xù)多年的使用,播娘蒿已對(duì)苯磺隆產(chǎn)生了嚴(yán)重抗藥性。本研究以采自北京、河南、河北和山東省份的麥田播娘蒿為研究試材,測(cè)定了不同播娘蒿種群對(duì)苯磺隆的抗性水平和抗性機(jī)理,將具有不同乙酰乳酸合成酶(ALS)突變型播娘蒿種群繁殖、純化為具有單一突變類型的種群,并以此為研究對(duì)象進(jìn)一步研究了不同ALS突變型對(duì)ALS除草劑交互抗性、ALS酶功能和支鏈氨基酸含量的影響,探討了利用分子生物學(xué)技術(shù)鑒定抗性植株的快速檢測(cè)方法,主要研究結(jié)果如下:1.抗苯磺隆播娘蒿種群初步鑒定利用區(qū)分劑量法鑒定了 73個(gè)播娘蒿種群對(duì)苯磺隆的抗性,結(jié)果表明:28個(gè)為抗性種群,18個(gè)種群具有潛在的抗性風(fēng)險(xiǎn),27個(gè)為敏感種群。2.抗性播娘蒿ALS基因突變多樣性研究播娘蒿ALS基因突變多樣性鑒定結(jié)果表明,播娘蒿體內(nèi)存在兩個(gè)ALS基因,分別為ALS1和 ALS2,ALS1 基因上 Pro197 突變成 Leu、Ser、Thr、His 或 Tyr,Asp376 突變成 Glu,Trp574突變成Leu,ALS2基因上Pro197突變成Ser或Thr。在28個(gè)抗性突變種群中Pro-197-Leu突變比例為 39.3%,Pro-197-Ser 突變比例為 28.6%,Pro-197-Thr 突變比例為 28.6%,Pro-197-His 突變比例為 10.7%,Pro-197-Tyr 突變比例為 3.6%,Asp-376-Glu 突變比例為 3.6%,Trp-574-Leu 突變比例為7.1%,其中Pro-197-Tyr突變?cè)诓ツ镙锷鲜鞘状舞b定并發(fā)現(xiàn)。Trp-574-Leu(in ALS1)突變和Pro-197-Thr(in ALS2)突變發(fā)生在同一抗性植株中,這是首次發(fā)現(xiàn)的在田間自然選擇條件下而形成的雙突變雜草。3.不同ALS突變型對(duì)播娘蒿抗性和交互抗性影響研究為了減少不同種群間遺傳背景差異,對(duì)具有不同ALS突變型的播娘蒿進(jìn)行單株繁殖,獲得了具有不同突變純合子的播娘蒿種群,分別為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)種群,以及 Trp-574-Leu(in ALS1)突變和 Pro-197-Thr(in ALS2)突變的 pHB30 種群。盆栽法測(cè)定了純化種群 pHB08、pHB22、pHB23、pHB24、pHB25、pHB42 和 pHB30 對(duì)苯磺隆的抗性水平,結(jié)果表明純化的播娘蒿對(duì)苯磺隆產(chǎn)生了高水平的抗性,其中pHB25(Asp-376-Glu)和 pHB30(Trp-574-Leu + Pro-197-Thr)種群比 pHB08(Pro-197-Leu)、pHB22(Pro-197-Ser)、pHB23(Pro-197-Thr)、pHB24(Pro-197-His)和 pHB42(Trp-574-Leu)種群對(duì)苯磺隆的抗性水平更高(789.3-815.0vs 152.3-366.3)。不同ALS突變型播娘蒿對(duì)ALS除草劑的交互抗性測(cè)定結(jié)果表明,Pro-197-Leu、Pro-197-Ser、Pro-197-Thr和Pro-197-His突變的播娘蒿種群對(duì)氯吡嘧磺隆(SU)、唑嘧磺草胺(TP)和氟唑磺隆(SCT)產(chǎn)生了交互抗性,對(duì)咪唑乙煙酸(IMI)和嘧啶肟草醚(PTB)沒有交互抗性;而Asp-376-Glu突變的pHB25種群,Trp-574-Leu突變的pHB42種群和雙突變的pHB30種群對(duì)氯吡嘧磺隆、唑嘧磺草胺、氟唑磺隆、咪唑乙煙酸和嘧啶肟草醚五類ALS除草劑都產(chǎn)生了交互抗性。4.不同ALS突變型對(duì)播娘蒿ALS功能影響研究乙酰乳酸合成酶催化活性和對(duì)苯磺隆敏感性測(cè)定結(jié)果表明,和敏感種群相比,抗性種群pHB08、pHB22、pHB23、pHB24、pHB25和pHB42乙酰乳酸合成酶對(duì)苯磺隆的敏感性分別下降了 250.0、205.8、263.7、708.4、2844.7 和 485.3 倍,且 pHB08、pHB22、pHB23 和 pHB24 種群ALS催化活性有顯著性降低(11%-21%),pHB25種群ALS催化活性有顯著性升高(26%),而pHB42種群ALS催化活性沒有顯著性差異。酶動(dòng)力學(xué)研究結(jié)果表明,和敏感種群相比,抗性種群pHB08、pHB22、pHB23、pHB24、pHB25和pHB42乙酰乳酸合成酶對(duì)底物的Km值和最大反應(yīng)速率Vmax值都有顯著性下降,Km值下降了0.63-0.87 倍,Vmax值下降了 0.30-0.91 倍。反饋抑制研究表明,Pro-197-Leu、Pro-197-Ser、Pro-197-Thr和Pro-197-His突變種群乙酰乳酸合成酶對(duì)亮氨酸的反饋抑制減弱,對(duì)纈氨酸和異亮氨酸的反饋抑制則沒有影響,Asp-376-Glu突變種群和Trp-574-Leu突變種群乙酰乳酸合成酶對(duì)亮氨酸、纈氨酸和異亮氨酸的反饋抑制都減弱。5.不同ALS突變型對(duì)播娘蒿體內(nèi)支鏈氨基酸含量影響不同ALS突變型播娘蒿體內(nèi)支鏈氨基酸含量測(cè)定結(jié)果表明,Pro-197-Leu、Pro-197-His、Pro-197-Ser和Pro-197-Thr突變抗性植株體內(nèi)亮氨酸和異亮氨酸含量和敏感植株沒有差異(Pro-197-Ser突變植株體內(nèi)異亮氨酸含量比敏感植株較低),Asp-376-Glu突變和Trp-574-Leu突變抗性植株體內(nèi)亮氨酸和纈氨酸含量比敏感植株顯著性增加。6.抗苯磺隆播娘蒿快速檢測(cè)方法研究針對(duì)敏感與抗性播娘蒿植株在ALS基因Pro197、Asp376或Trp574位點(diǎn)的堿基差異,用CAPS方法設(shè)計(jì)BsmFI、MboI和BsrDI酶切檢測(cè)了 48株播娘蒿植株的靶標(biāo)抗性,結(jié)果表明,此方法準(zhǔn)確率為100%,可用于快速檢測(cè)靶標(biāo)突變的抗性播娘蒿植株。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S451.2
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S451.2
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