氯虫苯甲酰胺干扰桃小食心虫交配的转录组分析

doi:10.3864/j.issn.0578-1752.2018.15.008

摘要/Abstract

摘要： 【目的】探明氯虫苯甲酰胺处理后桃小食心虫（Carposina sasakii）成虫的转录组差异，了解该药剂影响桃小食心虫交配的基因在功能分类和代谢通路等方面的生物学特征，挖掘与交配相关的功能基因。【方法】通过生物学实验观察氯虫苯甲酰胺干扰桃小食心虫成虫交配及繁殖情况。采用Illumina HiSeqTM2500高通量测序技术对刚羽化的桃小食心虫雌雄成虫、羽化后4—6 h进入交配高峰期的雌雄成虫和经氯虫苯甲酰胺处理4—6 h的雌雄成虫进行转录组测序，利用Trinity软件对所得序列进行de novo组装及评估，之后对获得的有效序列进行功能注释，并利用qRT-PCR技术分析氯虫苯甲酰胺处理后相关基因的时空表达变化。【结果】氯虫苯甲酰胺处理后，桃小食心虫的交配率显著降低，寿命缩短，产卵量减少。通过合并组装桃小食心虫转录组有效序列共获得102 831条unigene，其中34 526个有注释信息。根据筛选标准，氯虫苯甲酰胺处理过程中，雌雄虫中分别有122个和147个基因发生变化，其中相同差异基因31个。对所存在的234个差异基因进行GO功能注释和富集分析结果显示，分子功能过程中的催化活性和结合活性及生物学过程中与代谢过程、单一生物体过程和细胞过程相关的5类基因占主导地位。KEGG分类结果显示，富集到代谢通路的最多，有25个，包括昆虫激素合成、药物代谢等。通过比对分析，鉴定c64662.graph_c0为桃小食心虫鱼尼丁受体基因，其长度为15 637 bp，与已报道CsRyR的一致性为99.0%。此外，在234个差异基因中，鉴别羧酸酯酶unigene 3个、细胞色素P450 unigene 4个、肌钙蛋白unigene 3个、气味结合蛋白unigene 1个和生物钟unigene 1个。细胞色素P450 c40709.graph_c0参与昆虫激素生物合成。根据转录组中基因表达分析，12个基因在雌雄虫中的表达均出现不同变化趋势。qRT-PCR结果显示，氯虫苯甲酰胺诱导羧酸酯酶c51998.graph_c0基因上调表达；药剂处理后，雌雄虫的c57480.graph_c0和c53794.graph_c0的表达分别呈现显著的上调和下调变化，而c40709.graph_c0基因仅在雄虫中显著下调，处理6 h后c53281.graph_c0只在雌虫中上调表达；氯虫苯甲酰胺处理后3个肌钙蛋白基因在整个试验阶段均表现明显的下调趋势；雄虫中的生物钟unigene c60883.graph_c0和气味结合蛋白unigene c45675.graph_c0的变化趋势较为一致，进入暗期后立即上调，但均受氯虫苯甲酰胺的抑制。雌虫体内RyR的表达量显著上调，而雄虫初次到达求偶高峰期前RyR的表达量与对照差异不显著，到第2个求偶高峰期时，RyR表达显著下调。除细胞色素P450 c57480.graph_c0、c40709.graph_c0和c53281.graph_c0外，其余基因在雄虫中的表达均高于雌虫。且触角酯酶基因c54944.graph_c0、生物钟基因c60883.graph_c0和气味结合蛋白基因c45675.graph_c0在黑暗光照交替变化时，表达发生明显地上调或下调。【结论】通过转录组测序发现氯虫苯甲酰胺干扰桃小交配的作用机制是由靶标基因、嗅觉相关基因、代谢基因、生物钟基因等相互作用引起的。

关键词: 氯虫苯甲酰胺, 桃小食心虫, 交配, 干扰, 转录组

Abstract: 【Objective】 The objective of this study is to identify the difference of transcriptome of peach fruit moth (Carposina sasakii) adults under the stress of chlorantraniliprole, and describe biological characteristics involved in functional classifications and metabolic pathways, which is necessary to understand functional genes for mating. 【Method】Biological experiments were carried out to observe the effects of chlorantraniliprole on mating and reproduction of C. sasakii. The high throughput sequencing platform Illumina HiSeqTM2500 was used to sequence the transcriptome of C. sasakii adults (including virgin female and male, female and male with courtship behavior in the mating season after emergence for 4-6 h, female and male exposed to chlorantraniliprole for 4-6 h). De novo assembling and assessment were carried out by Trinity software, and then the effective sequential data were assigned to the relevant databases to perform functional annotation analysis. The temporal and spatial expression level of the related genes of C. sasakii was analyzed by qRT-PCR after chlorantraniliprole treatment.【Result】After chlorantraniliprole treatment, the mating rate, adult longevity and fertility of C. sasakii decreased. 102 831 unigenes were assembled from the transcriptomes, and 34 526 unigenes were annotated. According to the screening criteria, in the process of chlorantraniliprole treatment, 122 differentially expressed genes (DEGs) and 147 DEGs were identified in female and male adults, respectively, of which 31 DEGs were co-existed. The results of gene ontology (GO) functional annotation and enrichment analysis of 234 DEGs showed that these genes were annotated into 5 dominated process, including catalytic activity and binding activity in the molecular functional process, metabolic process, single-organism process and cellular process in the biological process. By KEGG pathways identification, 234 DEGs were enriched in 25 pathways for metabolic, which included insect hormone biosynthesis, drug metabolism, etc. The c64662.graph_c0 was identified as ryanodine receptor from C. sasakii by BLAST, its length is 15 637 bp and it has 99.0% identity with the reported CsRyR. Furthermore, 3 carboxylesterase unigenes, 4 cytochrome P450 unigenes, 3 troponin unigenes, 1 odorant binding protein unigene and 1 timeless unigene were annotated. The P450 c40709.graph_c0 is involved in insect hormone biosynthesis. Gene expression levels of 12 DEGs based on the FPKM value showed different trends. Finally, qRT-PCR was used to identify the 12 DEGs and RyR. The expression of carboxylesterase c51998.graph_c0 gene from C. sasakii adult was up-regulated after treated with chlorantraniliprole. The expression of cytochrome P450 c57480.graph_c0 and c53794.graph_c0 from C. sasakii female and male adults was significantly up-regulated and down-regulated, respectively, while the expression of cytochrome P450 c40709. graph_c0 gene was significantly down-regulated only in males, the expression of cytochrome P450 c53281.graph_c0 gene was up-regulated only in females after 6 h treatment. The expression of three troponin genes was significantly down-regulated during the whole trial period. The circadian clock unigene c60883.graph_c0 and odor binding protein unigene c45675.graph_c0 showed the same variation trend, which were up-regulated immediately after dark period, but were inhibited by chlorantraniliprole. The expression of RyR was significantly up-regulated in females, but there was no significant difference between male and control before the first courtship peak, and the expression of RyR was down-regulated significantly at the second courtship peak. The expression of other unigenes in the males was higher than that of the females except cytochrome P450s c57480.graph_c0, c40709.graph_c0 and c53281.graph_c0. Moreover, the expression of antennal esterase gene c54944.graph_c0, clock gene c60883.graph_c0 and odor binding protein gene c45675.graph_c0 was significantly up- or down- regulated when the dark/light changed alternately.【Conclusion】By transcriptome sequencing, it was found that the mechanism of disruption of mating in C. sasakii by chlorantraniliprole was possibly coursed by the interaction of target gene, olfactory related gene, metabolic gene, circadian clock gene and so on.

Key words: chlorantraniliprole, Carposina sasakii, mating, disruption, transcriptome

孙丽娜，田志强，张怀江，李艳艳，闫文涛，岳强，仇贵生. 氯虫苯甲酰胺干扰桃小食心虫交配的转录组分析[J]. 中国农业科学, 2018, 51(15): 2925-2936.

SUN LiNa, TIAN ZhiQiang, ZHANG HuaiJiang, LI YanYan, YAN WenTao, YUE Qiang, QIU GuiSheng. Transcriptome Analysis of Disruption of Mating in the Peach Fruit Moth (Carposina sasakii) by Chlorantraniliprole[J]. Scientia Agricultura Sinica, 2018, 51(15): 2925-2936.

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