棉铃虫烟碱型乙酰胆碱受体α7亚基基因克隆及功能研究
发布时间：2018-08-06 10:37
【摘要】：棉铃虫Helicoverpa armigera(Hübner)是我国棉花、玉米、小麦、等多种作物及花卉、蔬菜的重要害虫。Bt棉大面积推广种植对棉铃虫有较好的防控作用,但同时也增加了选择压,抗性风险增加。由于棉花生长后期Bt杀虫蛋白表达量下降,对三、四代棉铃虫控制作用降低,部分年份仍需喷洒化学农药进行防治。尽管Bt棉广泛种植减少了化学农药的使用,使棉铃虫对化学杀虫剂的抗性水平有所降低,但多年来抗性监测数据表明,棉铃虫对各种传统杀虫剂抗性仍保留在一定水平。乙基多杀菌素spinetoram是多杀菌素类杀虫剂的第二代产品,其活性是多杀菌素的10倍,对棉铃虫有较好的防治作用;既有化学农药的速效性,又有生物农药的安全性,而且作用机理新颖,与传统农药无交互抗性,对环境友好,低毒,无残留,符合新农药的发展方向,其作用机理的研究将有利于新农药的开发利用。前期对棉铃虫幼虫经乙基多杀菌素处理后差异表达转录组数据分析结果表明,烟碱型乙酰胆碱受体(n Ach Rs,nicotinic acetylcholine receptors)α7亚基m RNA表达量明显上调。因此本文克隆了棉铃虫n Ach Rsα7亚基基因,分析其表达谱,并对其功能进行了初步研究。利用简并引物和RACE(rapid-amplification of c DNA ends)技术成功获得了棉铃虫n Ach Rsα7亚基序列,该序列全长3632bp(Gen Bank登录号:KM884875),开放阅读框1491bp,编码496个氨基酸。具有半胱氨酸环受体家族的共同特征:细胞外N端和C端,四个跨膜区TM1、TM2、TM3、TM4,和一个大的胞内环;其中N端有配体结合区,有间隔13个氨基酸连接的两个半胱氨酸组成的半胱氨酸环,两个紧密相连的半胱氨酸;配体结合区和跨膜区保守性很高,胞内环变异性很大。与烟芽夜蛾的n Ach Rsα7-1核苷酸序列同源性99%,氨基酸序列同源性100%。通过构建进化树发现昆虫的n Ach Rsα5、α6、α7亚基与脊椎动物的α7亚基近缘。对昆虫的α5、α6、α7亚基进行氨基酸序列比对发现,胞内环区域有12个连续的氨基酸(RSSKSLLANVLD)完全相同,这是其它亚基没有的。表明昆虫的α5、α6、α7亚基可能具有某种类似的功能。利用实时荧光定量PCR(q RT-PCR)对棉铃虫n Ach Rsα7亚基m RNA在整个发育期、各组织间以及经乙基多杀菌素处理后的表达量进行了测定。结果发现:α7亚基m RNA在成虫表达量显著高于卵期,是卵期表达量的7.04倍;幼虫各龄期表达量较低,且无显著差异;幼虫头部表达量显著高于胸、腹部,是胸、腹部的5.42-6.04倍。经乙基多杀菌素LC60剂量处理72 h后n Ach Rsα7亚基表达量显著升高,是对照组的2.01倍。推测棉铃虫n Ach Rsα7亚基可能在乙基多杀菌素毒杀棉铃虫的过程中发挥一定的作用。利用两种si RNA(si RNA001,si RNA002)通过显微注射技术对棉铃虫3龄幼虫n Ach Rsα7亚基进行RNA干扰,通过q RT-PCR进行检测发现,72 h干扰效率分别为73.56%,66.39%,表明si RNA001干扰效果较好;si RNA001注射后对棉铃虫整个幼虫阶段体重变化没有显著影响;RNA干扰后,棉铃虫对乙基多杀菌素的敏感度升高,48 h死亡率和体重抑制率分别为36.11%,59.47%低于对照组52.78%,84.65%;72 h死亡率和体重抑制率分别为52.78%,65.24%低于对照组72.22%,90.18%%。表明棉铃虫n Ach Rsα7亚基是乙基多杀菌素潜在的作用靶标或是靶标受体的组成亚基。本文还对棉铃虫n Ach Rsα7亚基的体外表达进行了初步探索,将n Ach Rsα7亚基(c RNA)单独或是与鼠n Ach Rsβ2亚基共同以1:1的比例显微注射27.6 ng到爪蟾卵母细胞内,用乙酰胆碱刺激,通过双电极电压钳系统检测,发现都能检测到明显的电流变化,但是对同一个细胞用不同剂量刺激未能检测到连续的电流变化。以上结果表明,棉铃虫n Ach Rsα7亚基在乙基多杀菌素毒杀棉铃虫的过程中可能发挥一定的作用。通过构建爪蟾卵母细胞表达系统,可以进一步研究乙基多杀菌素与棉铃虫n Ach Rsα7亚基是否有相互作用以及药理特性,为以后研究n Ach Rs各亚基间的相互作用以及乙基多杀菌素的杀虫机理研究奠定基础。
[Abstract]:Cotton bollworm Helicoverpa armigera (H u BNER) is a variety of crops and flowers in China, such as cotton, corn, wheat, and other crops and flowers. The important pest of vegetable,.Bt cotton, has a good control effect on cotton bollworm, but it also increases the selection pressure and increases the risk of resistance. Because of the decline in the expression of Bt insecticidal protein in the late growth of cotton flower, it is three, four generation cotton. The control effect of bollworm was reduced, and chemical pesticides were still needed to prevent and control some years. Although extensive planting of Bt cotton decreased the use of chemical pesticides and reduced the resistance level of cotton bollworm to chemical insecticides, the resistance monitoring data for many years showed that the resistance of cotton bollworm to various traditional insecticides remained at a certain level. Bacteriocin spinetoram is the second generation of pasteicidal insecticide, its activity is 10 times of pasteicide, and it has good control effect on cotton bollworm. It has both the quick effect of chemical pesticides and the safety of biological pesticides, and the mechanism is novel. It has no interaction resistance with the traditional pesticide. It is friendly to environment, low toxicity and no residue, and conforms to new pesticide. The research of its mechanism of action will be beneficial to the development and utilization of new pesticides. The data analysis of the differential expression transcript of the larvae of Helicoverpa armigera after treatment with ethyl pasteurin showed that the expression of the nicotinic acetylcholine receptor (n Ach Rs, nicotinic acetylcholine receptors) alpha 7 subunit m RNA was obviously up-regulated. The gene of n Ach Rs alpha 7 subunit was augmated, and its expression profile was analyzed and its function was preliminarily studied. The sequence of n Ach Rs alpha 7 subunit of Helicoverpa armigera was obtained by degenerate primers and RACE (rapid-amplification of C DNA ends) technology. The sequence was full length, open reading frame and 496 amino groups. Acid. Having the common characteristics of the cysteine ring receptor family: the extracellular N and C terminals, four transmembrane regions TM1, TM2, TM3, TM4, and a large intracellular ring; the N terminal has a ligand binding zone, a cysteine ring consisting of two cysteine, which is connected by 13 amino acids, and two closely linked cysteine; ligand binding and transmembrane preservation The homology of the n Ach Rs alpha 7-1 nucleotide sequence of the moth Spodoptera Spodoptera was 99%. The amino acid sequence homologous 100%. found the n Ach Rs alpha 5, alpha 6, alpha 7 subunit and the alpha 7 subunit of vertebrates. The amino acid sequence alignment of the insect's alpha 5, alpha 6, and the alpha 7 subunit was found to be the inner ring region. 12 consecutive amino acids (RSSKSLLANVLD) are identical, which are not available to other subunits. It shows that the insect's alpha 5, alpha 6, alpha 7 subunit may have some similar function. The expression of n Ach Rs alpha 7 subunit m RNA at the whole development period, intertissue and ethyl pasteocidal by real-time fluorescent quantitative PCR (Q RT-PCR) The results showed that the expression of alpha 7 subunit m RNA was significantly higher than that in the egg stage, 7.04 times the egg stage expression, the expression of the larval age was lower and no significant difference. The expression of the larval head was significantly higher than that of the chest, abdomen, and the 5.42-6.04 times of the abdomen and the abdomen. The expression of n Ach Rs a 7 subunit after the treatment of sterotocidal LC60 was 72 h. The amount of n Ach Rs alpha 7 subunit of Helicoverpa armigera may play a role in the process of killing cotton bollworm with ethyl pasteusterin. Two kinds of Si RNA (Si RNA001, Si RNA002) were used to interfere with n Ach Rs alpha 7 subunit of 3 instar larvae of cotton bollworm, 7 2 h interference efficiency was 73.56%, 66.39%, indicating that Si RNA001 interference was better, Si RNA001 injection had no significant influence on the body weight change in the whole larval stage of cotton bollworm; after RNA interference, the sensitivity of cotton bollworm to ethyl pasteocidal, 48 h mortality and weight inhibition rate were 36.11%, 59.47% lower than the control group 52.78%, 84.65%; 72 h. The rate of death and weight inhibition was 52.78%, and 65.24% was lower than that of the control group. 90.18%%. showed that the n Ach Rs alpha 7 subunit of bollworm was a potential target for ethyl pasteocicin or the constituent subunit of the target receptor. This article also preliminarily explored the expression of n Ach Rs alpha 7 subunit of Helicoverpa armigera and the Ach Rs alpha 7 subunit of n Ach Rs (C RNA) alone or It was microinjected with n Ach Rs beta 2 subunit to microinjection of 27.6 ng into Xenopus oocyte in proportion of 1:1 and stimulated by acetylcholine and detected by two electrode voltage tweezers, it was found that the obvious current changes could be detected, but the same cell was not detected by different doses of the current. The n Ach Rs alpha 7 subunit may play a role in the killing of cotton bollworm by ethyl pasteusterin. By constructing the expression system of Xenopus oocyte, it is possible to further study the interaction and pharmacological properties of ethyl diocsterin and N Ach Rs alpha 7 subunit of cotton bollworm, and to study the interaction between Rs subunits of n Ach in the future. This study laid the foundation for the research of Insecticidal Mechanism of ethyl fungicides.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433
本文编号：2167468
[Abstract]:Cotton bollworm Helicoverpa armigera (H u BNER) is a variety of crops and flowers in China, such as cotton, corn, wheat, and other crops and flowers. The important pest of vegetable,.Bt cotton, has a good control effect on cotton bollworm, but it also increases the selection pressure and increases the risk of resistance. Because of the decline in the expression of Bt insecticidal protein in the late growth of cotton flower, it is three, four generation cotton. The control effect of bollworm was reduced, and chemical pesticides were still needed to prevent and control some years. Although extensive planting of Bt cotton decreased the use of chemical pesticides and reduced the resistance level of cotton bollworm to chemical insecticides, the resistance monitoring data for many years showed that the resistance of cotton bollworm to various traditional insecticides remained at a certain level. Bacteriocin spinetoram is the second generation of pasteicidal insecticide, its activity is 10 times of pasteicide, and it has good control effect on cotton bollworm. It has both the quick effect of chemical pesticides and the safety of biological pesticides, and the mechanism is novel. It has no interaction resistance with the traditional pesticide. It is friendly to environment, low toxicity and no residue, and conforms to new pesticide. The research of its mechanism of action will be beneficial to the development and utilization of new pesticides. The data analysis of the differential expression transcript of the larvae of Helicoverpa armigera after treatment with ethyl pasteurin showed that the expression of the nicotinic acetylcholine receptor (n Ach Rs, nicotinic acetylcholine receptors) alpha 7 subunit m RNA was obviously up-regulated. The gene of n Ach Rs alpha 7 subunit was augmated, and its expression profile was analyzed and its function was preliminarily studied. The sequence of n Ach Rs alpha 7 subunit of Helicoverpa armigera was obtained by degenerate primers and RACE (rapid-amplification of C DNA ends) technology. The sequence was full length, open reading frame and 496 amino groups. Acid. Having the common characteristics of the cysteine ring receptor family: the extracellular N and C terminals, four transmembrane regions TM1, TM2, TM3, TM4, and a large intracellular ring; the N terminal has a ligand binding zone, a cysteine ring consisting of two cysteine, which is connected by 13 amino acids, and two closely linked cysteine; ligand binding and transmembrane preservation The homology of the n Ach Rs alpha 7-1 nucleotide sequence of the moth Spodoptera Spodoptera was 99%. The amino acid sequence homologous 100%. found the n Ach Rs alpha 5, alpha 6, alpha 7 subunit and the alpha 7 subunit of vertebrates. The amino acid sequence alignment of the insect's alpha 5, alpha 6, and the alpha 7 subunit was found to be the inner ring region. 12 consecutive amino acids (RSSKSLLANVLD) are identical, which are not available to other subunits. It shows that the insect's alpha 5, alpha 6, alpha 7 subunit may have some similar function. The expression of n Ach Rs alpha 7 subunit m RNA at the whole development period, intertissue and ethyl pasteocidal by real-time fluorescent quantitative PCR (Q RT-PCR) The results showed that the expression of alpha 7 subunit m RNA was significantly higher than that in the egg stage, 7.04 times the egg stage expression, the expression of the larval age was lower and no significant difference. The expression of the larval head was significantly higher than that of the chest, abdomen, and the 5.42-6.04 times of the abdomen and the abdomen. The expression of n Ach Rs a 7 subunit after the treatment of sterotocidal LC60 was 72 h. The amount of n Ach Rs alpha 7 subunit of Helicoverpa armigera may play a role in the process of killing cotton bollworm with ethyl pasteusterin. Two kinds of Si RNA (Si RNA001, Si RNA002) were used to interfere with n Ach Rs alpha 7 subunit of 3 instar larvae of cotton bollworm, 7 2 h interference efficiency was 73.56%, 66.39%, indicating that Si RNA001 interference was better, Si RNA001 injection had no significant influence on the body weight change in the whole larval stage of cotton bollworm; after RNA interference, the sensitivity of cotton bollworm to ethyl pasteocidal, 48 h mortality and weight inhibition rate were 36.11%, 59.47% lower than the control group 52.78%, 84.65%; 72 h. The rate of death and weight inhibition was 52.78%, and 65.24% was lower than that of the control group. 90.18%%. showed that the n Ach Rs alpha 7 subunit of bollworm was a potential target for ethyl pasteocicin or the constituent subunit of the target receptor. This article also preliminarily explored the expression of n Ach Rs alpha 7 subunit of Helicoverpa armigera and the Ach Rs alpha 7 subunit of n Ach Rs (C RNA) alone or It was microinjected with n Ach Rs beta 2 subunit to microinjection of 27.6 ng into Xenopus oocyte in proportion of 1:1 and stimulated by acetylcholine and detected by two electrode voltage tweezers, it was found that the obvious current changes could be detected, but the same cell was not detected by different doses of the current. The n Ach Rs alpha 7 subunit may play a role in the killing of cotton bollworm by ethyl pasteusterin. By constructing the expression system of Xenopus oocyte, it is possible to further study the interaction and pharmacological properties of ethyl diocsterin and N Ach Rs alpha 7 subunit of cotton bollworm, and to study the interaction between Rs subunits of n Ach in the future. This study laid the foundation for the research of Insecticidal Mechanism of ethyl fungicides.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433
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