PR-XP1基因的克隆及在C6/36耐药细胞株中抗性的初步研究
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摘要
蚊虫作为全球广泛分布的最主要的医药害虫,传播多种致命疾病,严重威胁人类的健康。目前防控蚊虫危害的主要手段仍是化学防治;其中又以拟除虫菊酯类杀虫剂的使用最为广泛;然而其大量、长期的使用已使蚊虫产生了越来越严重的抗药性。因此,探明蚊虫针对拟除虫菊酯类杀虫剂的抗性机理,尤其抗性相关基因及其功能的研究就是非常必要的。长期以来,由于研究对象的复杂性以及研究技术的限制,目前对淡色库蚊氯菊酯抗性相关基因及其功能仍知之甚少。为了探索抗性淡色库蚊中全新的氯菊酯抗性相关基因并在细胞水平初步研究其抗性功能,本课题以抗性品系淡色库蚊与蚊虫C6/36细胞为研究对象,开展相关工作。
首先,采用了异硫氰酸胍法、Trizol法、Trizol改良法、Trizol+ CTAB法与CTAB法五种不同RNA提取方法对适合抗性品系淡色库蚊总RNA提取的最佳方法进行了探讨。通过对RNA纯度、浓度检测以及利用荧光实时定量PCR技术对不同基因的模板绝对值的监测验证,初步明确了本实验室首创Trizol + CTAB法是从淡色库蚊中提取总RNA的最佳方法,可以为淡色库蚊杀虫剂抗性相关基因克隆及其后续研究提供了新的模板获取技术。
其次,根据库蚊抗性相关EST(NCBI登录号为EC093826.1)设计基因特异性引物(GSP),利用了SMART-RACE技术,克隆出了目的基因5’/3’-末端序列,并进一步拼接、克隆得到了cDNA全长序列;再使用NCBI ORF finder软件定位其开放阅读框。结果显示,这是一条cDNA全长为2004 bp的全新基因,将其命名为PR-XP1,其编码蛋白为249个氨基酸。而后通过同源性比对、分子系统进化树构建、编码蛋白理化特性预测、疏水性及跨膜区预测、信号肽位点预测、磷酸化位点分析等生物信息软件的运用,对PR-XP1基因及其编码蛋白进行了评估与预测。结果表明,PR-XP1基因属于一个昆虫中独有的短序列“Hypothetic”蛋白基因家族;该基因编码蛋白具有特异的U型结构疏水性跨膜区,且具有很多磷酸化位点,因而推测其功能与杀虫剂抗性相关。其抗性相关也进一步被荧光实时定量PCR(相对定量)的监测结果所证实:PR-XP1基因在抗性品系蚊中表达量高于敏感品中的1.11倍。
第三,蚊虫抗性相关基因的功能研究无论是利用基因沉默技术还是转基因诱导方法,其细胞水平研究都必须有一个适合的抗性模型。基于此,我们采用MTT法检测了氯菊酯对蚊虫C6/36细胞杀伤作用(半数致死浓度为517.64μg/ml);并根据该数据计算后,利用逐步冲击法成功构建了在含300μg/ml氯菊酯的培养液中稳定生长及传代的耐药细胞株,并且还将继续筛选。最后,利用半定量RT-PCR方法检测了此前刚克隆获得的PR-XP1基因在该耐药细胞株中得相对表达量为普通细胞株的1.7倍以上。
综上所述,本研究为在淡色库蚊中研究抗性基因提供了总RNA提取的最佳方法;克隆了氯菊酯抗性相关基因PR-XP1并分析了相关的生物信息,以及监测了其抗性超量表达现象;并初步建立了蚊虫C6/36氯菊酯耐药细胞株以及首次验证了抗性相关基因在该耐药细胞株中的表达差异,为相关抗性基因功能的研究和蚊虫抗药性机制的探明提供了重要线索。
As the most widespread medical pest, mosquitoes could spread many deadly diseases, which threat human health seriously. Currently, chemical control is still the the primary way of mosquito control; and among them, Pyrethroid insecticides are used widely. However, mosquitos have formed more and more resistance to Pyrethroids for long-term and far-ranging using. Therefore, it is is very necessary to investigate the mechanism of resistance, expecially resistance related genes to pyrethroids. Because of the limited research strategy and technology, currently the permethrin-resistant genes and their functions of Culex pipiens pallens are still far to clarified. In order to investigate the new permethrin-resistant genes of Culex pipiens pallens and explore their Resistant function in cell, this study use resistant strain Culex pipiens pallens and mosquito C6/36 cell to carried out related work.
Firstly, using five different method of guanidinium isothiocyanate, Trizol, advanced Trizol, Trizol plus CTAB, and CTAB to isloate the total RNA from Culex pipiens pallens, we have got a best strategy to isolate total RNA——our results suggested that the method of Trizol plus CTAB, invented by our lab——which provides a good technology for further researchin.
Secondly, according to one Culex Pyrethroid-Resistance relevant EST(NCBI No. EC093826.1),we designed GSPs and used SMART-RACE technology to clone the gene 5’and 3’terminal sequence. And we tried to get the full length cDNA sequence by splicing, and after that we use NCBI ORF finder to position its open reading frame. The results showed that the full-length cDNA is 2004 bp, named the gene with PR-XP1, which encoding 249 amino acids. Additionally, bioinformatics software analysis, such as homologous comparison, phylogenetic tree construction, physical and chemical prop erties of protein-coding prediction, signal peptide sites prediction, phosphorylation site analysis and also on all suggested that gene PR-XP1 belongs to a more conservative“hypothetical protein”gene family,which only expressed in insects. The gene encodes a protein with a specific structure of U-hydrophobic transmembrane region, and has many phosphorylation sites; which revealed us to speculate that the function this gene is associated with insecticide resistance.And this was confirmed by the result of FQ-PCR monitoring, which showed that the expression level of PR-XP1 in resistant strains of mosquitoes was higher than 1.11 times in the sensitive strains.
Thirdly, suitable cell model is necessary in studying the function of mosquito pesticide-resistance genes, in whether depending on the method of transgenic gene or virus induced gene silencing. Based on this, we used the MTT assay to detect the killing effect of permethrin on mosquito C6/36 cells(Lc50=517.64μg/ml), which could grow and passage stably in nutrient solution with the conentration of Pyrethroid is 300μg/ml, and further constructed resistant cell lines. Last, we used semi-quantitative RT-PCR to detect the expression level of resistant gene of PR-XP1, which increased more than 1.7 time in resistant cell lines to the control group .
In summary, this study investigated the best strategy of total RNA extraction in Culex pipiens pallens; cloned permethrin-resistance related genes of PR-XP1 and analyzed by bioinformations, and then monitored the excessly expressional phenomenon. Based on these, this study further established mosquito C6/36 drug-resistant cell line to permethrin, and verified different expression level of resistance-related genes for the first time.And all of this provide an important clue for the studying and clarifying the mechanism of mosquito resistante gene.
首先,采用了异硫氰酸胍法、Trizol法、Trizol改良法、Trizol+ CTAB法与CTAB法五种不同RNA提取方法对适合抗性品系淡色库蚊总RNA提取的最佳方法进行了探讨。通过对RNA纯度、浓度检测以及利用荧光实时定量PCR技术对不同基因的模板绝对值的监测验证,初步明确了本实验室首创Trizol + CTAB法是从淡色库蚊中提取总RNA的最佳方法,可以为淡色库蚊杀虫剂抗性相关基因克隆及其后续研究提供了新的模板获取技术。
其次,根据库蚊抗性相关EST(NCBI登录号为EC093826.1)设计基因特异性引物(GSP),利用了SMART-RACE技术,克隆出了目的基因5’/3’-末端序列,并进一步拼接、克隆得到了cDNA全长序列;再使用NCBI ORF finder软件定位其开放阅读框。结果显示,这是一条cDNA全长为2004 bp的全新基因,将其命名为PR-XP1,其编码蛋白为249个氨基酸。而后通过同源性比对、分子系统进化树构建、编码蛋白理化特性预测、疏水性及跨膜区预测、信号肽位点预测、磷酸化位点分析等生物信息软件的运用,对PR-XP1基因及其编码蛋白进行了评估与预测。结果表明,PR-XP1基因属于一个昆虫中独有的短序列“Hypothetic”蛋白基因家族;该基因编码蛋白具有特异的U型结构疏水性跨膜区,且具有很多磷酸化位点,因而推测其功能与杀虫剂抗性相关。其抗性相关也进一步被荧光实时定量PCR(相对定量)的监测结果所证实:PR-XP1基因在抗性品系蚊中表达量高于敏感品中的1.11倍。
第三,蚊虫抗性相关基因的功能研究无论是利用基因沉默技术还是转基因诱导方法,其细胞水平研究都必须有一个适合的抗性模型。基于此,我们采用MTT法检测了氯菊酯对蚊虫C6/36细胞杀伤作用(半数致死浓度为517.64μg/ml);并根据该数据计算后,利用逐步冲击法成功构建了在含300μg/ml氯菊酯的培养液中稳定生长及传代的耐药细胞株,并且还将继续筛选。最后,利用半定量RT-PCR方法检测了此前刚克隆获得的PR-XP1基因在该耐药细胞株中得相对表达量为普通细胞株的1.7倍以上。
综上所述,本研究为在淡色库蚊中研究抗性基因提供了总RNA提取的最佳方法;克隆了氯菊酯抗性相关基因PR-XP1并分析了相关的生物信息,以及监测了其抗性超量表达现象;并初步建立了蚊虫C6/36氯菊酯耐药细胞株以及首次验证了抗性相关基因在该耐药细胞株中的表达差异,为相关抗性基因功能的研究和蚊虫抗药性机制的探明提供了重要线索。
As the most widespread medical pest, mosquitoes could spread many deadly diseases, which threat human health seriously. Currently, chemical control is still the the primary way of mosquito control; and among them, Pyrethroid insecticides are used widely. However, mosquitos have formed more and more resistance to Pyrethroids for long-term and far-ranging using. Therefore, it is is very necessary to investigate the mechanism of resistance, expecially resistance related genes to pyrethroids. Because of the limited research strategy and technology, currently the permethrin-resistant genes and their functions of Culex pipiens pallens are still far to clarified. In order to investigate the new permethrin-resistant genes of Culex pipiens pallens and explore their Resistant function in cell, this study use resistant strain Culex pipiens pallens and mosquito C6/36 cell to carried out related work.
Firstly, using five different method of guanidinium isothiocyanate, Trizol, advanced Trizol, Trizol plus CTAB, and CTAB to isloate the total RNA from Culex pipiens pallens, we have got a best strategy to isolate total RNA——our results suggested that the method of Trizol plus CTAB, invented by our lab——which provides a good technology for further researchin.
Secondly, according to one Culex Pyrethroid-Resistance relevant EST(NCBI No. EC093826.1),we designed GSPs and used SMART-RACE technology to clone the gene 5’and 3’terminal sequence. And we tried to get the full length cDNA sequence by splicing, and after that we use NCBI ORF finder to position its open reading frame. The results showed that the full-length cDNA is 2004 bp, named the gene with PR-XP1, which encoding 249 amino acids. Additionally, bioinformatics software analysis, such as homologous comparison, phylogenetic tree construction, physical and chemical prop erties of protein-coding prediction, signal peptide sites prediction, phosphorylation site analysis and also on all suggested that gene PR-XP1 belongs to a more conservative“hypothetical protein”gene family,which only expressed in insects. The gene encodes a protein with a specific structure of U-hydrophobic transmembrane region, and has many phosphorylation sites; which revealed us to speculate that the function this gene is associated with insecticide resistance.And this was confirmed by the result of FQ-PCR monitoring, which showed that the expression level of PR-XP1 in resistant strains of mosquitoes was higher than 1.11 times in the sensitive strains.
Thirdly, suitable cell model is necessary in studying the function of mosquito pesticide-resistance genes, in whether depending on the method of transgenic gene or virus induced gene silencing. Based on this, we used the MTT assay to detect the killing effect of permethrin on mosquito C6/36 cells(Lc50=517.64μg/ml), which could grow and passage stably in nutrient solution with the conentration of Pyrethroid is 300μg/ml, and further constructed resistant cell lines. Last, we used semi-quantitative RT-PCR to detect the expression level of resistant gene of PR-XP1, which increased more than 1.7 time in resistant cell lines to the control group .
In summary, this study investigated the best strategy of total RNA extraction in Culex pipiens pallens; cloned permethrin-resistance related genes of PR-XP1 and analyzed by bioinformations, and then monitored the excessly expressional phenomenon. Based on these, this study further established mosquito C6/36 drug-resistant cell line to permethrin, and verified different expression level of resistance-related genes for the first time.And all of this provide an important clue for the studying and clarifying the mechanism of mosquito resistante gene.
引文
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