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埃及伊蚊(Aedes aegypti)是虫媒病的主要传播媒介之一,雌蚊通过吸血传播登革热病毒、基孔肯亚病毒、寨卡病毒和黄热病病毒等多种病毒[1-2],严重危害人类的健康。2015年CDC报告指出,蚊虫在全球范围内造成了近一亿虫媒病病例,有效的蚊虫防控有利于公共卫生与健康。目前蚊虫防控主要包括环境防控、物理防控、化学防控和生物防控等方法,其中又主要依赖化学防控[3]。化学防控具有高效、简单、广谱等优点,常见的有拟除虫菊酯类、氨基甲酸酯类、有机磷、有机氯[4]、伊维菌素[5]和多杀菌素[6-7]等,但随着杀虫剂的大量及不合理使用,导致蚊虫对杀虫剂产生严重的抗性[8-11],探索新型杀虫剂对蚊虫防控具有重要意义。天维菌素(Tenvermectin,TVM)是一种新型十六元大环内酯类杀虫剂,是从基因工程菌链霉菌MHJ1011的发酵液中分离纯化的代谢产物。通过对阿维菌素产生菌(S. avermitilis)与米尔贝霉素(S. hygroscopicus)产生菌进行基因工程改造,从其代谢物中分离得到TVM A和TVM B两种有效成分[12]。有研究表明,天维菌素对小菜蛾(Plutella xylostella)、粘棉铃虫(Plutella xylostella)、松材线虫(Bursaphelenchus xylophilus)、朱砂叶螨(Tetranychus cinnabarinus)、柑橘红蜘蛛(Panonychus citri)和二斑叶螨(Tetranychus urticae)均有较高的杀虫活性[13-14]。本实验室的研究结果证明了大环内酯类抗寄生虫药伊维菌素、多杀菌素等对埃及伊蚊有非常好的杀灭作用[15];本实验室的前期实验结果表明,天维菌素对埃及伊蚊也具有一定的杀灭作用,但尚不清楚其作用机制。本研究以埃及伊蚊敏感株为实验材料,使用天维菌素处理埃及伊蚊幼虫,利用转录组测序并结合生物信息学分析,在转录组水平上比较埃及伊蚊幼虫在给药前后的基因表达差异,并利用Real-time Quantitative PCR(rt-qPCR)对差异表达基因进行验证。本研究在转录组水平上探究天维菌素对埃及伊蚊幼虫的作用机制,旨在为深入研究埃及伊蚊对天维菌素的抗性机制奠定基础。
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供试埃及伊蚊幼虫由海南大学生命科学与药学院热带动物医学与媒介生物学实验室提供。将20 g·L−1天维菌素用二甲基亚砜(DMSO)梯度稀释为200 mg·L−1稀释液,然后将1 mL天维菌素稀释液加入至99 mL的过夜去氯水里使天维菌素处理液的终浓度为2 mg·L−1(T),同时在对照组(Control)中加入1 mL二甲基亚砜,每处理组设3次重复。将20只3龄末4龄初的埃及伊蚊幼虫放入盛有天维菌素处理液或对照组处理液的杯中,处理24 h后统计并移除死亡幼虫,收集存活蚊虫用于下游转录组分析。
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采用Trizol法[16]提取药物处理组和对照组幼虫的总RNA,用1%的琼脂糖凝胶电泳检测,并用微量核酸检测仪测RNA浓度和纯度。利用Nanodrop及Bioanalyzer进行RNA质量验证。
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用带有Oligo(dT)的磁珠富集有polyA尾巴的mRNA,然用DNA探针杂交rRNA,去除rRNA纯化后即得到所需RNA。以获得的片段化RNA为模板,用随机的N6引物进行反转录得到cDNA,将cDNA末端补平,5′端磷酸化,3′段连接接头后进行PCR,PCR产物热变性成单链,再用一段桥式引物将单链DNA环化得到单链环状DNA文库,测序由华大基因有限公司完成。获得的转录组数据利用生物信息学方法进行数据过滤,基因组比对,差异表达分析及基因注释等。
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选取6个差异表达基因(Cuticle protein 19,Tetraspanin-11-like,Glycine-rich cell wall structural,Cuticle protein,Glutathione S-transferase-1,Carboxylesterase 2),利用Primer 5软件设计定量PCR引物(表1),以RPS17为内参基因,每个样品设3个重复,利用2−ΔΔCt法计算基因表达量。
表 1 荧光定量PCR引物
Table 1. Primers for fluorescence quantitative PCR
基因Gene 正向引物Forward primer 方向引物Reverse primer Cuticle protein 19 GGGGATGTCGTTAAGGGAG TTGTGGTGGTCGGATTTG Tet raspanin-11-like TGGACCGTAGTGGATAAGAA GTCGCAATCAGCACATAGA Glycine-rich cell wall structural CGATAGGTGGACAGGGAC AAACTGTGGACCGAAAGG Cuticle protein TCTTGTAGCTGCTCCACTCA TTCCTGCTGGGACTTCTG Glutathione S-transferase-1 AAGCCGAAGAGCACAAGA GACTCCACCAGGTAGACCA Carboxylesterase 2 GCAAAGCGATGAACATAA TACTGGTTGAACGGGACT Ribosomal protein S17 AAGAAGTGGCCATCATTCCA GGTCTCCGGGTCGACTTC
Transcriptome Analysis of Aedes aegypti Larvae before and after Treatment with Tenvermectin
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摘要: 为探索新型大环内酯类杀虫剂天维菌素对蚊虫防控的应用前景,进一步了解天维菌素对蚊虫的作用机制,本研究利用转录组测序技术研究埃及伊蚊幼虫参与药物代谢及靶标等生物学过程的基因。利用高通量测序获取给药前后埃及伊蚊的差异表达基因,进一步进行Gene Ontology(GO)及KEGG富集分析,并利用Real-time Quantitative PCR(rt-qPCR)验证目标基因表达情况。结果显示,与对照组相比,天维菌素处理后埃及伊蚊幼虫中有2 647个基因差异显著表达,其中上调基因有697个,下调表达的基因有1 950个。Gene Ontology分析显示,测序基因主要注释到细胞过程、代谢过程、膜、结合和催化活性等功能类。KEGG通路主要富集在药物代谢、免疫应答、生物合成以及消化与吸收等过程。选取GST-1,EST2等6个目标基因进行rt-qPCR验证,表达水平与测序结果一致。Abstract: Transcriptomes of Aedes aegypti larvae before and after administration of tenvermectin were sequenced with RNA-Seq to investigate the gene expression differences of A. aegypti at the transcriptome level before and after the treatment. The gene expression differences of A. aegypti before and after the treatment were then analyzed, and verified by real-time quantitative PCR. The results showed that, compared with the control group, there were 2 647 genes that were significantly different in expression in the larvae of A. aegypti treated with tenvermectin, of which 697 were up-regulated and 1 950 down-regulated. Gene ontology analysis showed that the sequenced genes were mainly annotated into such functional groups as cellular process, metabolic process, membrane, and binding and catalytic activity. The differential gene KEGG pathway is mainly enriched in medicine metabolism, immune response, biosynthesis, digestion and absorption. Six genes including three upregulated genes GST-1, TS11 and CarE2 and three downregulated genes CP, CP19 and GCW were randomly selected from the genes of the larvae treated, and verified by using real-time quantitative PCR, and the results were consistent with the RNA Seq results. RNA Seq was used to screen out the genes that had different expression of A. aegypti before and after treatment with tenvermectin, which provides insight into understanding of the toxicological mechanism of tenvermectin in A. aegypti and finding of the medicine resistance related genes from A. aegypti.
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Key words:
- tenvermectin /
- Aedes aegypti /
- RNA-Seq
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图 2 埃及伊蚊幼虫差异表达基因数量的分析
A为差异基因表达量的统计图;B为差异基因表达量的火山图;C为差异基因表达量的聚类热图,横坐标轴表示样本的log2(FPKM+1),纵坐标轴表示基因。
Fig. 2 Analysis of the quantity of differentially expressed genes
A.The statistical map of differential gene expression; B. The volcanic map of differential gene expression; C. The clustering heat map of differential gene expression. The horizontal axis represents the log2 (FPKM + 1) of the sample, and the vertical axis represents the gene.
表 1 荧光定量PCR引物
Table 1 Primers for fluorescence quantitative PCR
基因Gene 正向引物Forward primer 方向引物Reverse primer Cuticle protein 19 GGGGATGTCGTTAAGGGAG TTGTGGTGGTCGGATTTG Tet raspanin-11-like TGGACCGTAGTGGATAAGAA GTCGCAATCAGCACATAGA Glycine-rich cell wall structural CGATAGGTGGACAGGGAC AAACTGTGGACCGAAAGG Cuticle protein TCTTGTAGCTGCTCCACTCA TTCCTGCTGGGACTTCTG Glutathione S-transferase-1 AAGCCGAAGAGCACAAGA GACTCCACCAGGTAGACCA Carboxylesterase 2 GCAAAGCGATGAACATAA TACTGGTTGAACGGGACT Ribosomal protein S17 AAGAAGTGGCCATCATTCCA GGTCTCCGGGTCGACTTC -
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