马铃薯甲虫谷胱甘肽转移酶家族基因的鉴定及其对三种杀虫剂的响应
发布时间：2018-08-14 11:45
【摘要】：马铃薯甲虫是取食马铃薯叶片的重要害虫,常引起马铃薯产量的巨大损失。长期的化学杀虫剂使用导致抗药性快速发展,这使防治难度变得更大。研究发现,谷胱甘肽转移酶(glugathione S-transferase,GST)活性增强导致马铃薯甲虫幼虫对伏杀硫磷及氯菊酯的抗性。我们之前增效剂测定实验也表明,GST可能参与多种杀虫剂的解毒代谢。目前,马铃薯甲虫的GST基因家族尚未系统鉴定,参与杀虫剂代谢的GST基因也尚未明确。有鉴于此,本文主要开展了以下3个方面的工作。一、马铃薯甲虫GST基因挖掘及鉴定基于马铃薯甲虫的基因组和转录组数据,经过同源搜索和基因预测,发现34个GST候选序列。采用RT-PCR验证了上述候选GST基因序列的正确性,应用RACE技术获得了部分GST基因的全长cDNA序列(GenBank登录号为KU522306-KU522339)。结合赤拟谷盗、黑腹果蝇、家蚕、西方蜜蜂、冈比亚按蚊和豌豆蚜的GST蛋白,建立了系统发育树,分析了它们的系统进化。最终,我们获得了 30个GST基因,其中29个基因编码细胞质GST,1个基因编码微粒体GST。29个细胞质基质基因中,3、10、5、4、4、1 个基因分属于 delta、epsilon、omega、sigma、theta 和 zeta 亚家族。另外2个GST基因不属于已知亚家族而未能归类。二、马铃薯甲虫GST基因结构分析通过对GST基因进行系统发育及结构分析,我们发现,30个GST基因分布在17个染色体骨架(scaffolds)上。10个epsilon亚家族的GST基因中,有8个(LdGSTe1至LdGSTe8)分布在染色体骨架12上,2个(LdGST9和LdGSTe10)分布在染色体骨架1223上。亚家族epsilon的GST基因在染色体上的位置与系统发育树中预测的进化过程是一致的。另外,马铃薯甲虫中2个omega亚家族和1个sigma亚家族GST基因位于染色体骨架134。3个sigma亚家族GST基因位于scaffold 521。2个未归类GST基因位于染色体骨架615。其他GST基因则分别位于不同的染色体骨架。还分析了马铃薯甲虫GST基因的内含子-外显子结构。30个GST基因共包含83个内含子。平均每个GST基因包含2.8个内含子。不存在不包含内含子的基因。另外,我们还发现LdGSTe2、LdGST4和LdGSTo3存在多个可变剪切体。三、三种杀虫剂(高效氯氰菊酯、氟虫腈、硫丹)诱导GSTs的表达动态经高效氯氰菊酯处理后存活的马铃薯甲虫,其LdGSTe2a、LdGSTe2b、LdGSTe10、LdGSTu2、LdGSTo5 和LdGSTt1表达水平分别上调 18.0、9.8、3.4、11.8、4.9 和 21.4倍。与此类似,氟虫腈处理后,LdGSTe2a、LdGSTe2b、LdGSTo5和LdGSTt1分别上调了 3.6、28.3、4.8和 6.6 倍。硫丹处理后,LdGSTd1、LdGSTe2a、LdGSTe2b、LdGSTu2、LdGSTo5和LdGSTt1的转录水平分别上调了 4.9、5.9、13.8、9.4、12.0和4.1倍。高效氯氰菊酯处理后,LdGSTe4和LdGSTe6的mRNA表达水平显著下降。氟虫腈处理明显降低了 LdGSTe4、LdGSTe6和LdGSTe10的转录水平。硫丹处理明显降低了LdGSTe4、LdGSTe6 和 LdGSTz1 的表达水平。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.32
本文编号：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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.32
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