棉鈴蟲煙堿型乙酰膽堿受體α7亞基基因克隆及功能研究
發(fā)布時(shí)間：2018-08-06 10:37
【摘要】：棉鈴蟲Helicoverpa armigera(Hübner)是我國棉花、玉米、小麥、等多種作物及花卉、蔬菜的重要害蟲。Bt棉大面積推廣種植對(duì)棉鈴蟲有較好的防控作用,但同時(shí)也增加了選擇壓,抗性風(fēng)險(xiǎn)增加。由于棉花生長后期Bt殺蟲蛋白表達(dá)量下降,對(duì)三、四代棉鈴蟲控制作用降低,部分年份仍需噴灑化學(xué)農(nóng)藥進(jìn)行防治。盡管Bt棉廣泛種植減少了化學(xué)農(nóng)藥的使用,使棉鈴蟲對(duì)化學(xué)殺蟲劑的抗性水平有所降低,但多年來抗性監(jiān)測數(shù)據(jù)表明,棉鈴蟲對(duì)各種傳統(tǒng)殺蟲劑抗性仍保留在一定水平。乙基多殺菌素spinetoram是多殺菌素類殺蟲劑的第二代產(chǎn)品,其活性是多殺菌素的10倍,對(duì)棉鈴蟲有較好的防治作用;既有化學(xué)農(nóng)藥的速效性,又有生物農(nóng)藥的安全性,而且作用機(jī)理新穎,與傳統(tǒng)農(nóng)藥無交互抗性,對(duì)環(huán)境友好,低毒,無殘留,符合新農(nóng)藥的發(fā)展方向,其作用機(jī)理的研究將有利于新農(nóng)藥的開發(fā)利用。前期對(duì)棉鈴蟲幼蟲經(jīng)乙基多殺菌素處理后差異表達(dá)轉(zhuǎn)錄組數(shù)據(jù)分析結(jié)果表明,煙堿型乙酰膽堿受體(n Ach Rs,nicotinic acetylcholine receptors)α7亞基m RNA表達(dá)量明顯上調(diào)。因此本文克隆了棉鈴蟲n Ach Rsα7亞基基因,分析其表達(dá)譜,并對(duì)其功能進(jìn)行了初步研究。利用簡并引物和RACE(rapid-amplification of c DNA ends)技術(shù)成功獲得了棉鈴蟲n Ach Rsα7亞基序列,該序列全長3632bp(Gen Bank登錄號(hào):KM884875),開放閱讀框1491bp,編碼496個(gè)氨基酸。具有半胱氨酸環(huán)受體家族的共同特征:細(xì)胞外N端和C端,四個(gè)跨膜區(qū)TM1、TM2、TM3、TM4,和一個(gè)大的胞內(nèi)環(huán);其中N端有配體結(jié)合區(qū),有間隔13個(gè)氨基酸連接的兩個(gè)半胱氨酸組成的半胱氨酸環(huán),兩個(gè)緊密相連的半胱氨酸;配體結(jié)合區(qū)和跨膜區(qū)保守性很高,胞內(nèi)環(huán)變異性很大。與煙芽夜蛾的n Ach Rsα7-1核苷酸序列同源性99%,氨基酸序列同源性100%。通過構(gòu)建進(jìn)化樹發(fā)現(xiàn)昆蟲的n Ach Rsα5、α6、α7亞基與脊椎動(dòng)物的α7亞基近緣。對(duì)昆蟲的α5、α6、α7亞基進(jìn)行氨基酸序列比對(duì)發(fā)現(xiàn),胞內(nèi)環(huán)區(qū)域有12個(gè)連續(xù)的氨基酸(RSSKSLLANVLD)完全相同,這是其它亞基沒有的。表明昆蟲的α5、α6、α7亞基可能具有某種類似的功能。利用實(shí)時(shí)熒光定量PCR(q RT-PCR)對(duì)棉鈴蟲n Ach Rsα7亞基m RNA在整個(gè)發(fā)育期、各組織間以及經(jīng)乙基多殺菌素處理后的表達(dá)量進(jìn)行了測定。結(jié)果發(fā)現(xiàn):α7亞基m RNA在成蟲表達(dá)量顯著高于卵期,是卵期表達(dá)量的7.04倍;幼蟲各齡期表達(dá)量較低,且無顯著差異;幼蟲頭部表達(dá)量顯著高于胸、腹部,是胸、腹部的5.42-6.04倍。經(jīng)乙基多殺菌素LC60劑量處理72 h后n Ach Rsα7亞基表達(dá)量顯著升高,是對(duì)照組的2.01倍。推測棉鈴蟲n Ach Rsα7亞基可能在乙基多殺菌素毒殺棉鈴蟲的過程中發(fā)揮一定的作用。利用兩種si RNA(si RNA001,si RNA002)通過顯微注射技術(shù)對(duì)棉鈴蟲3齡幼蟲n Ach Rsα7亞基進(jìn)行RNA干擾,通過q RT-PCR進(jìn)行檢測發(fā)現(xiàn),72 h干擾效率分別為73.56%,66.39%,表明si RNA001干擾效果較好;si RNA001注射后對(duì)棉鈴蟲整個(gè)幼蟲階段體重變化沒有顯著影響;RNA干擾后,棉鈴蟲對(duì)乙基多殺菌素的敏感度升高,48 h死亡率和體重抑制率分別為36.11%,59.47%低于對(duì)照組52.78%,84.65%;72 h死亡率和體重抑制率分別為52.78%,65.24%低于對(duì)照組72.22%,90.18%%。表明棉鈴蟲n Ach Rsα7亞基是乙基多殺菌素潛在的作用靶標(biāo)或是靶標(biāo)受體的組成亞基。本文還對(duì)棉鈴蟲n Ach Rsα7亞基的體外表達(dá)進(jìn)行了初步探索,將n Ach Rsα7亞基(c RNA)單獨(dú)或是與鼠n Ach Rsβ2亞基共同以1:1的比例顯微注射27.6 ng到爪蟾卵母細(xì)胞內(nèi),用乙酰膽堿刺激,通過雙電極電壓鉗系統(tǒng)檢測,發(fā)現(xiàn)都能檢測到明顯的電流變化,但是對(duì)同一個(gè)細(xì)胞用不同劑量刺激未能檢測到連續(xù)的電流變化。以上結(jié)果表明,棉鈴蟲n Ach Rsα7亞基在乙基多殺菌素毒殺棉鈴蟲的過程中可能發(fā)揮一定的作用。通過構(gòu)建爪蟾卵母細(xì)胞表達(dá)系統(tǒng),可以進(jìn)一步研究乙基多殺菌素與棉鈴蟲n Ach Rsα7亞基是否有相互作用以及藥理特性,為以后研究n Ach Rs各亞基間的相互作用以及乙基多殺菌素的殺蟲機(jī)理研究奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433
本文編號(hào)：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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S433
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