灰飛虱（Laodelphax striatellus）谷胱甘肽-S-轉(zhuǎn)移酶基因的分子克隆及表達(dá)量分析
發(fā)布時(shí)間：2018-11-15 08:44
【摘要】：灰飛虱Laodelphax striatellus(Fallen)是我國重要的水稻害蟲,在刺吸水稻等禾本科植物的同時(shí),還能傳播多種植物病毒病,嚴(yán)重影響我國水稻的產(chǎn)量。谷胱甘肽-S-轉(zhuǎn)移酶(Glutathione S-transferases,GSTs)是一種多功能的超家族酶系。GSTs被認(rèn)為在昆蟲體內(nèi)行使殺蟲劑解毒代謝的功能,與昆蟲的抗藥性形成緊密相關(guān)。因此,本文利用灰飛虱轉(zhuǎn)錄組數(shù)據(jù),搜索并克隆了灰飛虱谷胱甘肽-S-轉(zhuǎn)移酶基因序列,通過熒光定量PCR技術(shù)對三種灰飛虱抗性品系中的谷胱甘肽-S-轉(zhuǎn)移酶基因進(jìn)行了表達(dá)分析,篩選出可能參與殺蟲劑解毒代謝的灰飛虱谷胱甘肽-S--轉(zhuǎn)移酶。研究結(jié)果總結(jié)如下:1、灰飛虱谷胱甘肽-S-轉(zhuǎn)移酶基因的分子克隆和序列分析通過本地Blast的方法搜索灰飛虱轉(zhuǎn)錄組數(shù)據(jù),獲得了19條谷胱甘肽-S-轉(zhuǎn)移酶基因序列,去除2條冗余序列,剩余的17條序列中9條是已發(fā)表的灰飛虱GST基因,另外8條是新發(fā)現(xiàn)的灰飛虱GSTs。上述新發(fā)現(xiàn)的8條序列通過PCR克隆和驗(yàn)證后,分別命名為 LsGSTd2、LsGSTo2、LsGSTo3、LsGSTz2、LsGSTm1、LsGSTm2、LsGSTm3和 LsGSTm4。其中,LsGSTd2、LsGSTz2、LsGSTm1、LsGSTm2、LsGSTm3 和 LsGSTm4基因序列完整,而LsGSTo2和LsGSTo3基因的3'端缺失。利用3'RACE技術(shù)克隆了LsGSTo 和LsGSTo3基因的3'端序列,并驗(yàn)證了它們的全長。LsGSTo2全長為1158 bp,開放閱讀框1119 bp,編碼372個(gè)氨基酸;LsGSTo3全長為648bp,開放閱讀框636bp,編碼211個(gè)氨基酸。綜合已知的9條灰飛虱GST基因和本次新發(fā)現(xiàn)的8條GSTs,通過系統(tǒng)發(fā)育分析,發(fā)現(xiàn)這17條灰飛虱GST基因中:Delta亞家族有2個(gè),Epsilon亞家族有1個(gè),Omega亞家族有3個(gè),Sigma亞家族有3個(gè),Theta亞家族有1個(gè),Zeta亞家族有2個(gè),Microsomal亞家族有5個(gè)。2、谷耽甘肽-S-轉(zhuǎn)移酶在三個(gè)灰飛虱抗性品系中的表達(dá)量分析為了篩選出灰飛虱體內(nèi)參與殺蟲劑解毒代謝的谷胱甘肽-S-轉(zhuǎn)移酶,利用定量PCR技術(shù)檢測了在三個(gè)灰飛虱抗性品系(吡蟲淋抗性品系、毒死蜱抗性品系和溴氰菊酯抗性品系)中,新克隆的8個(gè)GSTs基因和文獻(xiàn)中已報(bào)道的9條GSTs基因的表達(dá)情況。結(jié)果表明:在抗吡蟲啉的灰飛虱品系中LsGSTd2、LsGSTm1、LsGSTo2、LsGSTe1和LsGSTz1基因的表達(dá)量顯著高于敏感品系,而LsGSTm4基因表達(dá)量顯著下調(diào)為敏感品系的0.3倍;在抗毒死蜱的灰飛虱品系中LsGSTo2的表達(dá)量顯著高于敏感品系,而LsGSTm4基因表達(dá)量下調(diào)為敏感品系的0.5倍;在抗溴氰菊酯的灰飛虱品系中LsGSTd2、LsGSTo2、LsGSTs1、LsGSTe1和LsGSTz1基因表達(dá)量明顯上調(diào)。從以上研究結(jié)果可以發(fā)現(xiàn):在灰飛虱抗性品系中上調(diào)表達(dá)的基因除了有Delta亞家族中的LsGSTd2和 Epsilon 亞家族中的 LsGSTe1外,還有 Omega、Sigma、Zeta 和 Microsomal亞家族的GSTs基因。綜上所述,本研究通過對GST基因的克隆和表達(dá)量的分析,篩選出了可能參與殺蟲劑解毒代謝的GSTs,為進(jìn)一步研究GST在殺蟲劑解毒代謝中的功能奠定了堅(jiān)實(shí)的基礎(chǔ)。
[Abstract]:Ash planthopper (Laodelphax striatellus (Fallen) is an important rice pest in China. It can also spread a variety of plant virus diseases while sucking rice and other gramineous plants, which seriously affects the yield of rice in China. Glutathione-S-transferase (GSTs) is a multifunctional superfamily enzyme system. GSTs is thought to play a role in detoxification and metabolism of insecticides in insects, which is closely related to the formation of insecticide resistance in insects. Therefore, the glutathione-S-transferase gene sequence of ash planthopper was searched and cloned using transcriptional data. The expression of glutathione-S-transferase gene in three resistant strains of fly planthopper was analyzed by fluorescence quantitative PCR, and glutathione-S-transferase, which may be involved in the detoxification metabolism of planthopper, was screened out. The results were summarized as follows: 1. Molecular cloning and sequence analysis of Glutathione-S-transferase gene of fly ash planthopper obtained 19 glutathione-S-transferase gene sequences by searching transcriptional data of ash planthopper by local Blast. After removing 2 redundant sequences, 9 of the remaining 17 sequences were published GST genes, and the other 8 were newly discovered GSTs.. The eight newly discovered sequences were cloned and verified by PCR and named LsGSTd2,LsGSTo2,LsGSTo3,LsGSTz2,LsGSTm1,LsGSTm2,LsGSTm3 and LsGSTm4., respectively. The sequence of LsGSTd2,LsGSTz2,LsGSTm1,LsGSTm2,LsGSTm3 and LsGSTm4 genes was complete, and the 3 '-terminal deletion of LsGSTo2 and LsGSTo3 genes. The 3'terminal sequences of LsGSTo and LsGSTo3 genes were cloned by 3'RACE technique, and their full length was confirmed. The full length of LsGSTo2 was 1158 bp, open reading frame 1119 bp, encoding 372 amino acids, and LsGSTo3 was 648 BP, open reading frame 636 BP, encoding 211 amino acids. Through phylogenetic analysis of 9 known GST genes and 8 newly discovered GSTs, it was found that there were 2 Delta subfamilies, 1 Epsilon subfamily and 3 Omega subfamilies. There are 3 Sigma subfamilies, 1 Theta subfamily, 2 Zeta subfamilies and 5 Microsomal subfamilies. Analysis of the expression of glutamate-S-transferase in three resistant strains of fly planthopper in order to screen glutathione-S-transferase involved in insecticide detoxification metabolism, The expression of 8 newly cloned GSTs genes and 9 reported GSTs genes in three resistant strains (chlorpyrifos, chlorpyrifos and deltamethrin resistant lines) were detected by quantitative PCR technique. The results showed that the expression of LsGSTd2,LsGSTm1,LsGSTo2,LsGSTe1 and LsGSTz1 genes in imidacloprid resistant strains was significantly higher than that in sensitive strains, while the expression of LsGSTm4 gene was significantly decreased by 0.3 times of that of sensitive strains. The expression of LsGSTo2 in chlorpyrifos resistant strains was significantly higher than that in sensitive strains, while the expression of LsGSTm4 gene was down-regulated by 0.5 times of that of susceptible strains. The expression of LsGSTd2,LsGSTo2,LsGSTs1,LsGSTe1 and LsGSTz1 genes was up-regulated in deltamethrin resistant strains. From the above results, it was found that the up-regulated genes expressed in the resistant strains of planthopper were not only LsGSTd2 in Delta subfamily and LsGSTe1 in Epsilon subfamily, but also GSTs gene in Omega,Sigma,Zeta and Microsomal subfamily. In conclusion, through the cloning and expression analysis of GST gene, the GSTs, which may be involved in the detoxification metabolism of insecticides was selected, which laid a solid foundation for the further study of the function of GST in the detoxification metabolism of insecticides.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S435.112.3
本文編號：2332800
[Abstract]:Ash planthopper (Laodelphax striatellus (Fallen) is an important rice pest in China. It can also spread a variety of plant virus diseases while sucking rice and other gramineous plants, which seriously affects the yield of rice in China. Glutathione-S-transferase (GSTs) is a multifunctional superfamily enzyme system. GSTs is thought to play a role in detoxification and metabolism of insecticides in insects, which is closely related to the formation of insecticide resistance in insects. Therefore, the glutathione-S-transferase gene sequence of ash planthopper was searched and cloned using transcriptional data. The expression of glutathione-S-transferase gene in three resistant strains of fly planthopper was analyzed by fluorescence quantitative PCR, and glutathione-S-transferase, which may be involved in the detoxification metabolism of planthopper, was screened out. The results were summarized as follows: 1. Molecular cloning and sequence analysis of Glutathione-S-transferase gene of fly ash planthopper obtained 19 glutathione-S-transferase gene sequences by searching transcriptional data of ash planthopper by local Blast. After removing 2 redundant sequences, 9 of the remaining 17 sequences were published GST genes, and the other 8 were newly discovered GSTs.. The eight newly discovered sequences were cloned and verified by PCR and named LsGSTd2,LsGSTo2,LsGSTo3,LsGSTz2,LsGSTm1,LsGSTm2,LsGSTm3 and LsGSTm4., respectively. The sequence of LsGSTd2,LsGSTz2,LsGSTm1,LsGSTm2,LsGSTm3 and LsGSTm4 genes was complete, and the 3 '-terminal deletion of LsGSTo2 and LsGSTo3 genes. The 3'terminal sequences of LsGSTo and LsGSTo3 genes were cloned by 3'RACE technique, and their full length was confirmed. The full length of LsGSTo2 was 1158 bp, open reading frame 1119 bp, encoding 372 amino acids, and LsGSTo3 was 648 BP, open reading frame 636 BP, encoding 211 amino acids. Through phylogenetic analysis of 9 known GST genes and 8 newly discovered GSTs, it was found that there were 2 Delta subfamilies, 1 Epsilon subfamily and 3 Omega subfamilies. There are 3 Sigma subfamilies, 1 Theta subfamily, 2 Zeta subfamilies and 5 Microsomal subfamilies. Analysis of the expression of glutamate-S-transferase in three resistant strains of fly planthopper in order to screen glutathione-S-transferase involved in insecticide detoxification metabolism, The expression of 8 newly cloned GSTs genes and 9 reported GSTs genes in three resistant strains (chlorpyrifos, chlorpyrifos and deltamethrin resistant lines) were detected by quantitative PCR technique. The results showed that the expression of LsGSTd2,LsGSTm1,LsGSTo2,LsGSTe1 and LsGSTz1 genes in imidacloprid resistant strains was significantly higher than that in sensitive strains, while the expression of LsGSTm4 gene was significantly decreased by 0.3 times of that of sensitive strains. The expression of LsGSTo2 in chlorpyrifos resistant strains was significantly higher than that in sensitive strains, while the expression of LsGSTm4 gene was down-regulated by 0.5 times of that of susceptible strains. The expression of LsGSTd2,LsGSTo2,LsGSTs1,LsGSTe1 and LsGSTz1 genes was up-regulated in deltamethrin resistant strains. From the above results, it was found that the up-regulated genes expressed in the resistant strains of planthopper were not only LsGSTd2 in Delta subfamily and LsGSTe1 in Epsilon subfamily, but also GSTs gene in Omega,Sigma,Zeta and Microsomal subfamily. In conclusion, through the cloning and expression analysis of GST gene, the GSTs, which may be involved in the detoxification metabolism of insecticides was selected, which laid a solid foundation for the further study of the function of GST in the detoxification metabolism of insecticides.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S435.112.3
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