灰飞虱（Laodelphax striatellus）谷胱甘肽-S-转移酶基因的分子克隆及表达量分析

发布时间：2018-11-15 08:44

【摘要】：灰飞虱Laodelphax striatellus(Fallen)是我国重要的水稻害虫,在刺吸水稻等禾本科植物的同时,还能传播多种植物病毒病,严重影响我国水稻的产量。谷胱甘肽-S-转移酶(Glutathione S-transferases,GSTs)是一种多功能的超家族酶系。GSTs被认为在昆虫体内行使杀虫剂解毒代谢的功能,与昆虫的抗药性形成紧密相关。因此,本文利用灰飞虱转录组数据,搜索并克隆了灰飞虱谷胱甘肽-S-转移酶基因序列,通过荧光定量PCR技术对三种灰飞虱抗性品系中的谷胱甘肽-S-转移酶基因进行了表达分析,筛选出可能参与杀虫剂解毒代谢的灰飞虱谷胱甘肽-S--转移酶。研究结果总结如下:1、灰飞虱谷胱甘肽-S-转移酶基因的分子克隆和序列分析通过本地Blast的方法搜索灰飞虱转录组数据,获得了19条谷胱甘肽-S-转移酶基因序列,去除2条冗余序列,剩余的17条序列中9条是已发表的灰飞虱GST基因,另外8条是新发现的灰飞虱GSTs。上述新发现的8条序列通过PCR克隆和验证后,分别命名为 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技术克隆了LsGSTo 和LsGSTo3基因的3'端序列,并验证了它们的全长。LsGSTo2全长为1158 bp,开放阅读框1119 bp,编码372个氨基酸;LsGSTo3全长为648bp,开放阅读框636bp,编码211个氨基酸。综合已知的9条灰飞虱GST基因和本次新发现的8条GSTs,通过系统发育分析,发现这17条灰飞虱GST基因中:Delta亚家族有2个,Epsilon亚家族有1个,Omega亚家族有3个,Sigma亚家族有3个,Theta亚家族有1个,Zeta亚家族有2个,Microsomal亚家族有5个。2、谷耽甘肽-S-转移酶在三个灰飞虱抗性品系中的表达量分析为了筛选出灰飞虱体内参与杀虫剂解毒代谢的谷胱甘肽-S-转移酶,利用定量PCR技术检测了在三个灰飞虱抗性品系(吡虫淋抗性品系、毒死蜱抗性品系和溴氰菊酯抗性品系)中,新克隆的8个GSTs基因和文献中已报道的9条GSTs基因的表达情况。结果表明:在抗吡虫啉的灰飞虱品系中LsGSTd2、LsGSTm1、LsGSTo2、LsGSTe1和LsGSTz1基因的表达量显著高于敏感品系,而LsGSTm4基因表达量显著下调为敏感品系的0.3倍;在抗毒死蜱的灰飞虱品系中LsGSTo2的表达量显著高于敏感品系,而LsGSTm4基因表达量下调为敏感品系的0.5倍;在抗溴氰菊酯的灰飞虱品系中LsGSTd2、LsGSTo2、LsGSTs1、LsGSTe1和LsGSTz1基因表达量明显上调。从以上研究结果可以发现:在灰飞虱抗性品系中上调表达的基因除了有Delta亚家族中的LsGSTd2和 Epsilon 亚家族中的 LsGSTe1外,还有 Omega、Sigma、Zeta 和 Microsomal亚家族的GSTs基因。综上所述,本研究通过对GST基因的克隆和表达量的分析,筛选出了可能参与杀虫剂解毒代谢的GSTs,为进一步研究GST在杀虫剂解毒代谢中的功能奠定了坚实的基础。
[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.
【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】：S435.112.3

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