淡色库蚊miRNA表达谱及其与抗药性关系研究
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摘要
蚊媒病是全世界重要的传染病之一，包括疟疾，西尼罗河热等严重危害人类健康的疾病。蚊媒病控制的主要措施是治理蚊媒。化学防治因其速杀、长效、方便等特点，一直是蚊媒综合治理策略中的主要方法。然而随着杀虫剂连续、大量的使用，蚊媒抗药性开始产生和发展。蚊媒抗药性产生和发展加剧了蚊媒病传播的恶化趋势。化学防治因其能够快速，有效控制蚊媒种群，在以后很长的时间内仍是蚊媒治理的主要方法。因此蚊媒抗药性研究仍是控制蚊媒病传播的重点。
蚊媒抗药性机制包括3个主要方面。一方面是靶标抗性，即蚊媒杀虫剂靶位点的改变；另一方面代谢抗性，其涉及代谢相关酶类转录水平的变化；此外为生理抗性，如表皮改变影响杀虫剂的穿透性。其中，以代谢抗性研究最多。前期代谢抗性研究关注杀虫剂代谢相关酶活性的改变，未见抗性相关基因转录后调控的研究。本实验初步探索了抗药性基因转录调控机制。
miRNA是一类转录后调控的重要因子，广泛存在于动物和植物中。其通过降低mRNA表达或者抑制蛋白质合成参与调控体内近30%的基因。miRNA功能包括调节生物体的生长、发育、凋亡等。目前在蚊中已经开始研究miRNA与抗感染方面的研究，至今未见miRNA和杀虫剂关系的报道。
本研究选取在我国分布广泛的重要家栖性病媒蚊种淡色库蚊为研究对象，以近年我国使用最多的卫生杀虫剂溴氰菊酯为材料，采用solexa测序法，比较了溴氰菊酯敏感和抗性淡色库蚊miRNA表达谱；通过PCR、T-A克隆及测序验证了pre-miRNA；采用定量PCR进一步验证了敏感和抗性品系中miRNA表达差异；采用双荧光报告验证了miR-71可以调控杀虫剂代谢酶类CYP325BG3表达。
本研究主要结果如下：
1通过WHO幼虫浸渍法建立了淡色库蚊溴氰菊酯抗性品系，其LC50为0.85mg/L，敏感品系LC50为0.03mg/L，抗性系数为28.3。
2使用solexa法对淡色库蚊溴氰菊酯敏感和抗性品系进行了成功的测序。送测序mRNA，敏感蚊混合样本浓度为670ug/ml, OD260/0D280为1.80；抗性蚊混合样本浓度为600ug/ml，OD260/0D280为1.83。经过琼脂糖凝胶电泳和安捷伦Bianalyzer检测，RNA完整性良好，测序结果中rRNA的比例小于40%。表明solexa测序结果良好，可以用于继续分析。
3对测序得到的原始数据进行去除接头、污染序列及低质量reads的处理，并统计sRNA序列长度分布，可以观测到20-23nt长度的序列所占比例高。将测序结果与最近缘致倦库蚊的基因组进行比对。将上述测序得到的序列（clean序列）按照rRNAetc> known miRNA> repeat> exon> intron的优先级顺序注释。在鉴定淡色库蚊miRNA的时候，根据致倦库蚊基因组序列，提取miRNA前体序列，进行二级结构分析。进一步用miRAlign软件将上述序列与miRBase比对，筛选出淡色库蚊成熟miRNA和预测新的miRNA。在淡色库蚊中共鉴定保守miRNA85个，前体结构91个；新miRNA143个，前体结构209个。同时比较了淡色库蚊敏感和抗性品系中miRNA表达量的差异。
4在淡色库蚊miRNA检测中，检测到miR-932、miR-980、miR-998、miR-999等昆虫特有miRNA；miR-1891、miR-1889和miR-2941等蚊特有miRNA。根据miRNA基因簇分析方法，设10K bp为miRNA基因之间排列的最大距离，共发现7个淡色库蚊miRNA簇，其中包括在昆虫中最常见的let-7、miR-125构成的miRNA簇。PCR鉴定了淡色库蚊pre-miRNA，T-A克隆测序了部分淡色库蚊pre-miRNA序列。测序结果显示，与致倦库蚊miRNA前体结构相比，淡色库蚊miRNA的茎部结构保守，而环部碱基突变比较常见。其为淡色库蚊和致倦库蚊的系统分类提供了一种新的可行性。
5选取solexa测序后比较有统计学差异的miRNA和昆虫学研究中已有功能报道miRNA进行定量验证。在敏感品系大部分发育阶段，miR-279-3p、miR-13、miR-989、miR-4448和miR-285的表达水平比抗性品系高；miR-317、miR-2b、miR-92a在抗性品系各发育阶段表达水平比敏感品系高。
6靶标预测提示，CYP325BG3的3’UTR序列和miR-71可能存在相互作用。采用双荧光报告实验证实，miR-71和CYP325BG3之间确实存在相互作用。结果表明，miR-71可能通过调节CYP325BG3的表达水平参与淡色库蚊溴氰菊酯抗性。
本研究首次报告了淡色库蚊抗药性miRNA表达谱，PCR和T-A克隆测序验证了miRNA前体结构，定量PCR鉴定了抗药性相关miRNA。双荧光报告方法验证miR-71可以调控代谢酶类CYP325BG3的表达，进而参与杀虫剂抗性。研究结果丰富了昆虫miRNA数据库，初步探索了miRNA在蚊抗药性中的作用，具有重要的理论意义和潜在的实际应用价值。
The mosquito-borne diseases including malaria, West Nile fever are importantinfectious diseases in the world and caused serious threat to human health. Mosquitocontrol is the main measure to combat mosquito-borne diseases. Chemical control,because of its killing speed, long-lasting and convenient features, has been the mainmethod in the comprehensive management strategy. Unfortunately, excessive andcontinuous usage of insecticides induced the development and spread of resistance.Then insecticide resistance exacerbated the spread of mosquito-borne diseases.However, after a very long time, chemical control for its rapid and efficientadvantage will still be the main method in control of mosquito-borne diseases.
Insecticide resistance mechanisms include three main areas: target resistance,that is, the change of the insecticide target sites. Metabolic resistance, that is, thechange of metabolic enzymes transcription level involved in metabolic resistance,and physiological resistance, such as cuticle protein and chitin enzyme also involvedin insecticide resistance. Preliminary studies concerned more about the change ofmetabolic enzyme activity, and there is no report on gene transcription regulation. Inthis study, we wanted to explore the resistance mechanism on regulate transcriptionlevel.
As an important factor on post-transcriptional regulation, miRNA was widelyfound in animals and plants. miRNA can regulate nearly30%genes throughdegradating mRNA expression or inhibiting protein synthesis. Organism's growth,development, and apoptosis can be regulated by miRNA. Recently, there were moreattentions on miRNA against infection in mosquito. So far there is no report on therelationship between miRNA and insecticide resistance.
This study selected Culex pipiens pallens (Cx. pipiens pallens) as the researchobject which is widely distributed in the northern China. We compared miRNA expression differences of deltamethrin-susceptible and–resistant strains by solexasequence. PCR and T-A cloning were used to verify pre-miRNA. Quantitative PCRfurther validated miRNA expression differences between resistant and susceptiblestrains. The dual fluorescence report test was used to verify miR-71can regulateCYP325BG3expression.
In this study, the main results are as follows:
1Two experimental strains were established in laboratory with WHO larvaebioassay method. LC50of the resistant strains is0.85mg/L and LC50of sensitivestrains is0.03mg/L. There is28.3times difference between the two strains.
2Solexa was used to sequence the miRNA of deltamethrin resistant andsusceptible strains. mRNA of the sensitive strain sample has the concentration670ug/ml and OD260/0D280is1.80. The resistant strain sample has the concentration670ug/ml and OD260/0D280is1.83. After detected with agarose gel electrophoresis andAgilent Bianalyzer, RNA integrity is good for solexa sequencing, and the ratio ofrRNA is less than40%, suggesting that solexa sequencing results can be used tocontinue analysis.
3After removing joints of the adaptors, pollution sequence and reads with lowquality from the raw data, we statisted sRNA length distribution and the sequence ofthe20-23nt length occupied high proportion through the distribution. The obtainedsequences (clean sequence) were annotated in accordance with rRNAetc> knownmiRNA> repeat> exon> intron. The miRNA precursor (pre-miRNA) of Cx. pipienspallens was extracted according to sequence of the genome of Cx. quinquefasciatusand the secondary structure was analysed. miRAlign software was further applied inthe above sequences and the sequences were aligned in miRBase.85conservedmiRNAs were identified and they have91precursors.143new miRNAs werescreened and they have209precursors in Cx. pipiens pallens. At the same time, we compared the miRNA difference between sensitive and resistant strains of Cx.pipiens pallens.
4We detected insect specific miRNAs, miR-932, miR-980, miR-998, miR-999and mosquito-specific miRNAs, miR-1891, miR-1889and miR-2941in our data.miRNA cluster was analysed with the10K bp as the maximum distance. Sevenclusters were found in our data, including let-7and miR-125cluster which is mostcommon in insects. PCR and T-A cloning were used to detect the pre-miRNA. Thesequencing results show that the stem of precursor is conserved, while the nucleotidemutation is more common in the loop compared with Cx. quinquefasciatus. Thisphenomenon provides a new feasibility for the systematic classification ofCx.pipiens pallens and Cx.quinquefascias.
5We selected significant different miRNA detected by solexa sequencing andfunction known miRNA for quantitative verification. The expression levels ofmiR-279-3p, miR-13, miR-989, miR-4448、miR-71and miR-285are higher in thesusceptible strain than in the resistant strain, and those of miR-317, miR-2b andmiR-92a are higher in the resistant strain than in the susceptible strain.
We find the interations of miR-71and CYP325BG33'UTR sequence throughtarget prediction principle. Dual fluorescence report test confirmed the interactionbetween miR-71and CYP325BG3. We speculate that miR-71may involve indeltamethrin resistance through regulating the expression level of CYP325BG3.
This study firstly reported the miRNA expression profiling in Cx. pipienspallens, identificated the differentially expressed miRNA in deltamethrin resistantand susceptible strains, and found miR-71can regulate CYP325BG3transcription.Our study has important theoretical significance and potential practical value.
蚊媒抗药性机制包括3个主要方面。一方面是靶标抗性，即蚊媒杀虫剂靶位点的改变；另一方面代谢抗性，其涉及代谢相关酶类转录水平的变化；此外为生理抗性，如表皮改变影响杀虫剂的穿透性。其中，以代谢抗性研究最多。前期代谢抗性研究关注杀虫剂代谢相关酶活性的改变，未见抗性相关基因转录后调控的研究。本实验初步探索了抗药性基因转录调控机制。
miRNA是一类转录后调控的重要因子，广泛存在于动物和植物中。其通过降低mRNA表达或者抑制蛋白质合成参与调控体内近30%的基因。miRNA功能包括调节生物体的生长、发育、凋亡等。目前在蚊中已经开始研究miRNA与抗感染方面的研究，至今未见miRNA和杀虫剂关系的报道。
本研究选取在我国分布广泛的重要家栖性病媒蚊种淡色库蚊为研究对象，以近年我国使用最多的卫生杀虫剂溴氰菊酯为材料，采用solexa测序法，比较了溴氰菊酯敏感和抗性淡色库蚊miRNA表达谱；通过PCR、T-A克隆及测序验证了pre-miRNA；采用定量PCR进一步验证了敏感和抗性品系中miRNA表达差异；采用双荧光报告验证了miR-71可以调控杀虫剂代谢酶类CYP325BG3表达。
本研究主要结果如下：
1通过WHO幼虫浸渍法建立了淡色库蚊溴氰菊酯抗性品系，其LC50为0.85mg/L，敏感品系LC50为0.03mg/L，抗性系数为28.3。
2使用solexa法对淡色库蚊溴氰菊酯敏感和抗性品系进行了成功的测序。送测序mRNA，敏感蚊混合样本浓度为670ug/ml, OD260/0D280为1.80；抗性蚊混合样本浓度为600ug/ml，OD260/0D280为1.83。经过琼脂糖凝胶电泳和安捷伦Bianalyzer检测，RNA完整性良好，测序结果中rRNA的比例小于40%。表明solexa测序结果良好，可以用于继续分析。
3对测序得到的原始数据进行去除接头、污染序列及低质量reads的处理，并统计sRNA序列长度分布，可以观测到20-23nt长度的序列所占比例高。将测序结果与最近缘致倦库蚊的基因组进行比对。将上述测序得到的序列（clean序列）按照rRNAetc> known miRNA> repeat> exon> intron的优先级顺序注释。在鉴定淡色库蚊miRNA的时候，根据致倦库蚊基因组序列，提取miRNA前体序列，进行二级结构分析。进一步用miRAlign软件将上述序列与miRBase比对，筛选出淡色库蚊成熟miRNA和预测新的miRNA。在淡色库蚊中共鉴定保守miRNA85个，前体结构91个；新miRNA143个，前体结构209个。同时比较了淡色库蚊敏感和抗性品系中miRNA表达量的差异。
4在淡色库蚊miRNA检测中，检测到miR-932、miR-980、miR-998、miR-999等昆虫特有miRNA；miR-1891、miR-1889和miR-2941等蚊特有miRNA。根据miRNA基因簇分析方法，设10K bp为miRNA基因之间排列的最大距离，共发现7个淡色库蚊miRNA簇，其中包括在昆虫中最常见的let-7、miR-125构成的miRNA簇。PCR鉴定了淡色库蚊pre-miRNA，T-A克隆测序了部分淡色库蚊pre-miRNA序列。测序结果显示，与致倦库蚊miRNA前体结构相比，淡色库蚊miRNA的茎部结构保守，而环部碱基突变比较常见。其为淡色库蚊和致倦库蚊的系统分类提供了一种新的可行性。
5选取solexa测序后比较有统计学差异的miRNA和昆虫学研究中已有功能报道miRNA进行定量验证。在敏感品系大部分发育阶段，miR-279-3p、miR-13、miR-989、miR-4448和miR-285的表达水平比抗性品系高；miR-317、miR-2b、miR-92a在抗性品系各发育阶段表达水平比敏感品系高。
6靶标预测提示，CYP325BG3的3’UTR序列和miR-71可能存在相互作用。采用双荧光报告实验证实，miR-71和CYP325BG3之间确实存在相互作用。结果表明，miR-71可能通过调节CYP325BG3的表达水平参与淡色库蚊溴氰菊酯抗性。
本研究首次报告了淡色库蚊抗药性miRNA表达谱，PCR和T-A克隆测序验证了miRNA前体结构，定量PCR鉴定了抗药性相关miRNA。双荧光报告方法验证miR-71可以调控代谢酶类CYP325BG3的表达，进而参与杀虫剂抗性。研究结果丰富了昆虫miRNA数据库，初步探索了miRNA在蚊抗药性中的作用，具有重要的理论意义和潜在的实际应用价值。
The mosquito-borne diseases including malaria, West Nile fever are importantinfectious diseases in the world and caused serious threat to human health. Mosquitocontrol is the main measure to combat mosquito-borne diseases. Chemical control,because of its killing speed, long-lasting and convenient features, has been the mainmethod in the comprehensive management strategy. Unfortunately, excessive andcontinuous usage of insecticides induced the development and spread of resistance.Then insecticide resistance exacerbated the spread of mosquito-borne diseases.However, after a very long time, chemical control for its rapid and efficientadvantage will still be the main method in control of mosquito-borne diseases.
Insecticide resistance mechanisms include three main areas: target resistance,that is, the change of the insecticide target sites. Metabolic resistance, that is, thechange of metabolic enzymes transcription level involved in metabolic resistance,and physiological resistance, such as cuticle protein and chitin enzyme also involvedin insecticide resistance. Preliminary studies concerned more about the change ofmetabolic enzyme activity, and there is no report on gene transcription regulation. Inthis study, we wanted to explore the resistance mechanism on regulate transcriptionlevel.
As an important factor on post-transcriptional regulation, miRNA was widelyfound in animals and plants. miRNA can regulate nearly30%genes throughdegradating mRNA expression or inhibiting protein synthesis. Organism's growth,development, and apoptosis can be regulated by miRNA. Recently, there were moreattentions on miRNA against infection in mosquito. So far there is no report on therelationship between miRNA and insecticide resistance.
This study selected Culex pipiens pallens (Cx. pipiens pallens) as the researchobject which is widely distributed in the northern China. We compared miRNA expression differences of deltamethrin-susceptible and–resistant strains by solexasequence. PCR and T-A cloning were used to verify pre-miRNA. Quantitative PCRfurther validated miRNA expression differences between resistant and susceptiblestrains. The dual fluorescence report test was used to verify miR-71can regulateCYP325BG3expression.
In this study, the main results are as follows:
1Two experimental strains were established in laboratory with WHO larvaebioassay method. LC50of the resistant strains is0.85mg/L and LC50of sensitivestrains is0.03mg/L. There is28.3times difference between the two strains.
2Solexa was used to sequence the miRNA of deltamethrin resistant andsusceptible strains. mRNA of the sensitive strain sample has the concentration670ug/ml and OD260/0D280is1.80. The resistant strain sample has the concentration670ug/ml and OD260/0D280is1.83. After detected with agarose gel electrophoresis andAgilent Bianalyzer, RNA integrity is good for solexa sequencing, and the ratio ofrRNA is less than40%, suggesting that solexa sequencing results can be used tocontinue analysis.
3After removing joints of the adaptors, pollution sequence and reads with lowquality from the raw data, we statisted sRNA length distribution and the sequence ofthe20-23nt length occupied high proportion through the distribution. The obtainedsequences (clean sequence) were annotated in accordance with rRNAetc> knownmiRNA> repeat> exon> intron. The miRNA precursor (pre-miRNA) of Cx. pipienspallens was extracted according to sequence of the genome of Cx. quinquefasciatusand the secondary structure was analysed. miRAlign software was further applied inthe above sequences and the sequences were aligned in miRBase.85conservedmiRNAs were identified and they have91precursors.143new miRNAs werescreened and they have209precursors in Cx. pipiens pallens. At the same time, we compared the miRNA difference between sensitive and resistant strains of Cx.pipiens pallens.
4We detected insect specific miRNAs, miR-932, miR-980, miR-998, miR-999and mosquito-specific miRNAs, miR-1891, miR-1889and miR-2941in our data.miRNA cluster was analysed with the10K bp as the maximum distance. Sevenclusters were found in our data, including let-7and miR-125cluster which is mostcommon in insects. PCR and T-A cloning were used to detect the pre-miRNA. Thesequencing results show that the stem of precursor is conserved, while the nucleotidemutation is more common in the loop compared with Cx. quinquefasciatus. Thisphenomenon provides a new feasibility for the systematic classification ofCx.pipiens pallens and Cx.quinquefascias.
5We selected significant different miRNA detected by solexa sequencing andfunction known miRNA for quantitative verification. The expression levels ofmiR-279-3p, miR-13, miR-989, miR-4448、miR-71and miR-285are higher in thesusceptible strain than in the resistant strain, and those of miR-317, miR-2b andmiR-92a are higher in the resistant strain than in the susceptible strain.
We find the interations of miR-71and CYP325BG33'UTR sequence throughtarget prediction principle. Dual fluorescence report test confirmed the interactionbetween miR-71and CYP325BG3. We speculate that miR-71may involve indeltamethrin resistance through regulating the expression level of CYP325BG3.
This study firstly reported the miRNA expression profiling in Cx. pipienspallens, identificated the differentially expressed miRNA in deltamethrin resistantand susceptible strains, and found miR-71can regulate CYP325BG3transcription.Our study has important theoretical significance and potential practical value.
引文
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