馬鈴薯甲蟲谷胱甘肽轉(zhuǎn)移酶家族基因的鑒定及其對三種殺蟲劑的響應(yīng)
發(fā)布時(shí)間：2018-08-14 11:45
【摘要】：馬鈴薯甲蟲是取食馬鈴薯葉片的重要害蟲,常引起馬鈴薯產(chǎn)量的巨大損失。長期的化學(xué)殺蟲劑使用導(dǎo)致抗藥性快速發(fā)展,這使防治難度變得更大。研究發(fā)現(xiàn),谷胱甘肽轉(zhuǎn)移酶(glugathione S-transferase,GST)活性增強(qiáng)導(dǎo)致馬鈴薯甲蟲幼蟲對伏殺硫磷及氯菊酯的抗性。我們之前增效劑測定實(shí)驗(yàn)也表明,GST可能參與多種殺蟲劑的解毒代謝。目前,馬鈴薯甲蟲的GST基因家族尚未系統(tǒng)鑒定,參與殺蟲劑代謝的GST基因也尚未明確。有鑒于此,本文主要開展了以下3個(gè)方面的工作。一、馬鈴薯甲蟲GST基因挖掘及鑒定基于馬鈴薯甲蟲的基因組和轉(zhuǎn)錄組數(shù)據(jù),經(jīng)過同源搜索和基因預(yù)測,發(fā)現(xiàn)34個(gè)GST候選序列。采用RT-PCR驗(yàn)證了上述候選GST基因序列的正確性,應(yīng)用RACE技術(shù)獲得了部分GST基因的全長cDNA序列(GenBank登錄號(hào)為KU522306-KU522339)。結(jié)合赤擬谷盜、黑腹果蠅、家蠶、西方蜜蜂、岡比亞按蚊和豌豆蚜的GST蛋白,建立了系統(tǒng)發(fā)育樹,分析了它們的系統(tǒng)進(jìn)化。最終,我們獲得了 30個(gè)GST基因,其中29個(gè)基因編碼細(xì)胞質(zhì)GST,1個(gè)基因編碼微粒體GST。29個(gè)細(xì)胞質(zhì)基質(zhì)基因中,3、10、5、4、4、1 個(gè)基因分屬于 delta、epsilon、omega、sigma、theta 和 zeta 亞家族。另外2個(gè)GST基因不屬于已知亞家族而未能歸類。二、馬鈴薯甲蟲GST基因結(jié)構(gòu)分析通過對GST基因進(jìn)行系統(tǒng)發(fā)育及結(jié)構(gòu)分析,我們發(fā)現(xiàn),30個(gè)GST基因分布在17個(gè)染色體骨架(scaffolds)上。10個(gè)epsilon亞家族的GST基因中,有8個(gè)(LdGSTe1至LdGSTe8)分布在染色體骨架12上,2個(gè)(LdGST9和LdGSTe10)分布在染色體骨架1223上。亞家族e(cuò)psilon的GST基因在染色體上的位置與系統(tǒng)發(fā)育樹中預(yù)測的進(jìn)化過程是一致的。另外,馬鈴薯甲蟲中2個(gè)omega亞家族和1個(gè)sigma亞家族GST基因位于染色體骨架134。3個(gè)sigma亞家族GST基因位于scaffold 521。2個(gè)未歸類GST基因位于染色體骨架615。其他GST基因則分別位于不同的染色體骨架。還分析了馬鈴薯甲蟲GST基因的內(nèi)含子-外顯子結(jié)構(gòu)。30個(gè)GST基因共包含83個(gè)內(nèi)含子。平均每個(gè)GST基因包含2.8個(gè)內(nèi)含子。不存在不包含內(nèi)含子的基因。另外,我們還發(fā)現(xiàn)LdGSTe2、LdGST4和LdGSTo3存在多個(gè)可變剪切體。三、三種殺蟲劑(高效氯氰菊酯、氟蟲腈、硫丹)誘導(dǎo)GSTs的表達(dá)動(dòng)態(tài)經(jīng)高效氯氰菊酯處理后存活的馬鈴薯甲蟲,其LdGSTe2a、LdGSTe2b、LdGSTe10、LdGSTu2、LdGSTo5 和LdGSTt1表達(dá)水平分別上調(diào) 18.0、9.8、3.4、11.8、4.9 和 21.4倍。與此類似,氟蟲腈處理后,LdGSTe2a、LdGSTe2b、LdGSTo5和LdGSTt1分別上調(diào)了 3.6、28.3、4.8和 6.6 倍。硫丹處理后,LdGSTd1、LdGSTe2a、LdGSTe2b、LdGSTu2、LdGSTo5和LdGSTt1的轉(zhuǎn)錄水平分別上調(diào)了 4.9、5.9、13.8、9.4、12.0和4.1倍。高效氯氰菊酯處理后,LdGSTe4和LdGSTe6的mRNA表達(dá)水平顯著下降。氟蟲腈處理明顯降低了 LdGSTe4、LdGSTe6和LdGSTe10的轉(zhuǎn)錄水平。硫丹處理明顯降低了LdGSTe4、LdGSTe6 和 LdGSTz1 的表達(dá)水平。
[Abstract]:Potato beetle is an important pest feeding on potato leaves, which often results in great loss of potato yield. Long-term use of chemical insecticides leads to rapid development of resistance, which makes control more difficult. It was found that the increased activity of glugathione S-transferase (glugathione S-transferase) resulted in the resistance of potato beetle larvae to fenthion and permethrin. Our previous synergist assay also suggested that GST may be involved in the detoxification metabolism of many insecticides. At present, the GST gene family of potato beetles has not been systematically identified, and the GST gene involved in pesticide metabolism has not been identified. In view of this, this paper mainly carries out the following three aspects of work. Firstly, the GST gene of potato beetle was mined and identified. Based on the genomic and transcriptional data of potato beetle, 34 GST candidate sequences were found by homology search and gene prediction. The correctness of the candidate GST gene sequence was verified by RT-PCR, and the full-length cDNA sequence of part of GST gene (GenBank accession number was KU522306-KU522339) was obtained by RACE technique. The phylogenetic tree was established and their phylogeny was analyzed by combining the GST proteins of Artemisia albopictus, Drosophila melanogaster, Bombyx mori, western bee, Anopheles gambiae and pea aphid. Finally, we obtained 30 GST genes, including 29 genes encoding cytoplasmic GSTs, 1 gene encoding microsomal GST.29 cytoplasmic genes, 1 gene belonging to the delta-epsilon omega sigmatheta and zeta subfamilies. The other two GST genes did not belong to the known subfamily and could not be classified. Secondly, the structural analysis of GST gene of potato beetle, through the phylogenetic and structural analysis of GST gene, we found that 30 GST genes were distributed in GST genes of 10 epsilon subfamilies on 17 chromosomal skeleton (scaffolds). Eight (LdGSTe1 to LdGSTe8) were found on chromosome skeleton 12 and two (LdGST9 and LdGSTe10) on chromosome skeleton 1223. The position of GST gene of subfamily epsilon on chromosome is consistent with the evolutionary process predicted in phylogenetic tree. In addition, in potato beetles, two omega subfamilies and one sigma subfamily GST gene were located in the chromosome skeleton 134.3 sigma subfamily GST gene located in scaffold 521.2 unclassified GST gene located in chromosome skeleton 615. Other GST genes are located in different chromosomal skeletons. The intron-exon structure of potato beetle GST gene was also analyzed. The 30 GST genes contained 83 introns. The average GST gene contains 2.8 introns. There is no gene that does not contain introns. In addition, we also found that there are many variable shear bodies in LdGSTe2LdGST4 and LdGSTo3. Three, three insecticides (high efficiency cypermethrin, fluronitrile, endosulfan) induced the expression dynamics of GSTs in potato beetles treated with high efficiency cypermethrin. The expression levels of LdGSTe2bT10LdGSTe10LdGSTu2LdGSTo5 and LdGSTu2LdGSTo5 and LdGSTe10LdGSTu2LdGSTo5 and LdGSTe10LdGSTo5 and LdGSTo5 were up-regulated by 18.09.83.411.84.and 21.4 times, respectively. Similarly, LdGSTo5 and LdGSTt1 of LdGSTe2aGSTo5 and LdGSTe2bGSTo5 were increased by 3.6% and 6.6 times respectively after treatment with fluronitrile. After endosulfan treatment, the transcription levels of LdGSTd1, LdGSTe2aAN, LdGSTe2bGSTo5 and LdGSTu2LdGSTo5, LdGSTo5 and LdGSTu2LdGSTo5 were increased by 4.1-fold and 4.1-fold, respectively. The mRNA expression of LdGSTe4 and LdGSTe6 decreased significantly after treatment with high efficiency cypermethrin. Fluronitrile treatment significantly reduced the transcription levels of LdGSTe4, LdGSTe6 and LdGSTe10. Endosulfan treatment significantly reduced the expression of LdGSTe4, LdGSTe6 and LdGSTz1.
【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(hào)】：S435.32
本文編號(hào)：2182758
[Abstract]:Potato beetle is an important pest feeding on potato leaves, which often results in great loss of potato yield. Long-term use of chemical insecticides leads to rapid development of resistance, which makes control more difficult. It was found that the increased activity of glugathione S-transferase (glugathione S-transferase) resulted in the resistance of potato beetle larvae to fenthion and permethrin. Our previous synergist assay also suggested that GST may be involved in the detoxification metabolism of many insecticides. At present, the GST gene family of potato beetles has not been systematically identified, and the GST gene involved in pesticide metabolism has not been identified. In view of this, this paper mainly carries out the following three aspects of work. Firstly, the GST gene of potato beetle was mined and identified. Based on the genomic and transcriptional data of potato beetle, 34 GST candidate sequences were found by homology search and gene prediction. The correctness of the candidate GST gene sequence was verified by RT-PCR, and the full-length cDNA sequence of part of GST gene (GenBank accession number was KU522306-KU522339) was obtained by RACE technique. The phylogenetic tree was established and their phylogeny was analyzed by combining the GST proteins of Artemisia albopictus, Drosophila melanogaster, Bombyx mori, western bee, Anopheles gambiae and pea aphid. Finally, we obtained 30 GST genes, including 29 genes encoding cytoplasmic GSTs, 1 gene encoding microsomal GST.29 cytoplasmic genes, 1 gene belonging to the delta-epsilon omega sigmatheta and zeta subfamilies. The other two GST genes did not belong to the known subfamily and could not be classified. Secondly, the structural analysis of GST gene of potato beetle, through the phylogenetic and structural analysis of GST gene, we found that 30 GST genes were distributed in GST genes of 10 epsilon subfamilies on 17 chromosomal skeleton (scaffolds). Eight (LdGSTe1 to LdGSTe8) were found on chromosome skeleton 12 and two (LdGST9 and LdGSTe10) on chromosome skeleton 1223. The position of GST gene of subfamily epsilon on chromosome is consistent with the evolutionary process predicted in phylogenetic tree. In addition, in potato beetles, two omega subfamilies and one sigma subfamily GST gene were located in the chromosome skeleton 134.3 sigma subfamily GST gene located in scaffold 521.2 unclassified GST gene located in chromosome skeleton 615. Other GST genes are located in different chromosomal skeletons. The intron-exon structure of potato beetle GST gene was also analyzed. The 30 GST genes contained 83 introns. The average GST gene contains 2.8 introns. There is no gene that does not contain introns. In addition, we also found that there are many variable shear bodies in LdGSTe2LdGST4 and LdGSTo3. Three, three insecticides (high efficiency cypermethrin, fluronitrile, endosulfan) induced the expression dynamics of GSTs in potato beetles treated with high efficiency cypermethrin. The expression levels of LdGSTe2bT10LdGSTe10LdGSTu2LdGSTo5 and LdGSTu2LdGSTo5 and LdGSTe10LdGSTu2LdGSTo5 and LdGSTe10LdGSTo5 and LdGSTo5 were up-regulated by 18.09.83.411.84.and 21.4 times, respectively. Similarly, LdGSTo5 and LdGSTt1 of LdGSTe2aGSTo5 and LdGSTe2bGSTo5 were increased by 3.6% and 6.6 times respectively after treatment with fluronitrile. After endosulfan treatment, the transcription levels of LdGSTd1, LdGSTe2aAN, LdGSTe2bGSTo5 and LdGSTu2LdGSTo5, LdGSTo5 and LdGSTu2LdGSTo5 were increased by 4.1-fold and 4.1-fold, respectively. The mRNA expression of LdGSTe4 and LdGSTe6 decreased significantly after treatment with high efficiency cypermethrin. Fluronitrile treatment significantly reduced the transcription levels of LdGSTe4, LdGSTe6 and LdGSTe10. Endosulfan treatment significantly reduced the expression of LdGSTe4, LdGSTe6 and LdGSTz1.
【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(hào)】：S435.32
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