美国白蛾嗅觉基因的鉴定及表达研究

发布时间：2018-08-16 16:59

【摘要】：昆虫在漫长的进化过程中形成了高度灵敏和极其复杂的嗅觉系统,这种专一的嗅觉感受机制通过感受、识别外界环境各种特定的化学气味,从而参与到昆虫的多种生理活动中,如选择寄主、找寻配偶、寻觅产卵所、觅食以及趋避等。而昆虫性信息素和各种植物挥发物质作为化学通讯的中介,在昆虫的嗅觉感受过程起到了重要的作用。美国白蛾Hyphantria cunea(Drury),是一种世界性检疫害虫,其爆发性的蔓延与扩散使我国遭受无法估量的经济损失和巨大的生态灾难。美国白蛾已成为我国最重要的外来入侵种之一,防控形势十分严峻。当前,国内外学者对于昆虫嗅觉机制和化学感受基因的研究都集中于分泌Type-Ⅰ型性信息素的鳞翅目害虫上,而产生Type-II型性信息素—美国白蛾的嗅觉分子机制未见报道。因此,迫切需要对美国白蛾进行嗅觉机制尤其是性信息素通讯的分子机制的相关研究,从而为发掘美国白蛾科学防控路径提供新的思路。本实验采用高通量测序技术对美国白蛾雌雄触角进行转录组测序;接着,利用生物信息学方法发掘和鉴定嗅觉相关基因。在此基础上,进一步通过组织表达分析,对相关基因的表达谱进行研究。主要结果如下:1.美国白蛾触角转录组测序及生物信息学分析:(1)美国白蛾触角转录组测序产生的碱基数约为8G,共获得64,020,776个Clean reads。经Trinity拼接组装后得到78,131条转录本(Transcripts),最终筛选得到59,243条Unigenes。有15,242个Unigenes可以同源比对到Nr数据库其他物种中,其中美国白蛾与家蚕(Bombyx mori)比对的序列最多,达48.1%,其次是黑脉金斑蝶(Danaus plexippus,29.5%)。(2)转录组数据中,12,565个Unigenes成功进行GO注释,5,781个Unigenes成功进行KO注释。(3)美国白蛾触角转录组59,243个Unigenes中,表达量较高的(FPKM10)基因7708个。表达丰度最高的50个基因中,7个是OBP(Odorant binding protein),2个是CSP(Chemosensory protein)。2.美国白蛾嗅觉基因的鉴定与表达:(1)对转录组数据进行同源比对,鉴定得到30个气味结合蛋白(OBP)基因,17个化学感受蛋白(CSP)基因,52个气味受体(OR)基因,10个羧酸酯酶(CXE)基因,2个感觉神经元膜蛋白(SNMP)基因,9个味觉受体(GR)基因和10个离子型受体(IR)基因。(2)通过反转录PCR(RT-PCR)和荧光定量PCR(RT-qPCR)对部分嗅觉基因进行组织表达研究,结果表明:部分OBP表现出触角高表达;3个PBP(Pheromone binding protein)为雄性触角高表达;CSP具有广泛的组织表达谱;HyphOR27(ORco)在触角表达量较高,3个PR(HyphOR1,HyphOR7,HyphOR50)雄性触角高表达;CXE在幼虫上广泛表达,在雌雄触角上各有高表达的基因;HyphSNMP1和HyphSNMP2分别在雌、雄触角上表达量高。本研究首次对美国白蛾触角进行了转录组分析,鉴定获得了美国白蛾嗅觉相关基因;同时,明确了主要嗅觉基因在雌雄个体间、不同组织间的表达特异性;该研究为明确美国白蛾性信息素通讯的分子机制奠定了基础,也为探索美国白蛾嗅觉分子控制新途径提供了重要依据。另外,本研究对于其它Type-Ⅱ型性信息素蛾类昆虫的嗅觉感受机制研究具有重要借鉴意义,拓展了鳞翅目蛾类嗅觉分子机制的研究。
[Abstract]:Insects have developed a highly sensitive and complex olfactory system during their long evolutionary process. This unique olfactory sensing mechanism recognizes various specific chemical odors in the external environment through sensation, thus participating in a variety of physiological activities of insects, such as host selection, mate search, spawning, foraging and avoidance. Sexual pheromones and plant volatiles play an important role in the olfactory process of insects. Hyphantria cunea (Drury), a worldwide quarantine pest, suffers immeasurable economic losses and enormous ecological disasters due to its explosive spread. Nowadays, the research on olfactory mechanism and chemosensitivity genes of insects has been concentrated on Lepidoptera pests secreting type-I sex pheromone, but the molecular mechanism of type-II sex pheromone, the olfactory mechanism of American white moth, has not been reported. Therefore, it is urgent to study the olfactory mechanism, especially the molecular mechanism of sex pheromone communication, in order to provide new ideas for the scientific prevention and control of white moth. The main results are as follows: (1) Sequencing and bioinformatics analysis of the antennal transcriptome of the American white moth, Hyphantria xylostella, yielded 64,020,776 Clean reads. After splicing and assembling, 78,131 transcripts (Transcripts) were obtained, and 59,243 Unigenes were screened out. 15,242 Unigenes could be homologously compared with other species in Nr database. Among them, the sequence of Bombyx Mori and White Moth (Bombyx mori) was 48.1%, followed by Danaus plexippus (29.5%). 12,565 Unigenes were successfully annotated with GO, and 5,781 Unigenes were successfully annotated with KO. (3) Of 59,243 Unigenes in the antennal transcription group, 7708 genes were highly expressed (FPKM10). Identification and expression: (1) Homologous comparison of transcriptome data identified 30 odor-binding protein (OBP) genes, 17 chemoreceptor protein (CSP) genes, 52 odor receptor (OR) genes, 10 carboxylesterase (CXE) genes, 2 sensory neuron membrane protein (SNMP) genes, 9 taste receptor (GR) genes and 10 ionotype receptor (IR) genes. (2) Reverse transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative PCR (RT-qPCR) were used to study the tissue expression of some olfactory genes. The results showed that some OBPs were highly expressed in antennae, three PBPs (Pheromone binding protein) were highly expressed in male antennae, CSP had extensive tissue expression profiles, HyphOR27 (ORco) was highly expressed in antennae, and three PR (HyphOR1, Hyp ORco) were highly expressed in antennae. HOR7, HyphOR50) is highly expressed in male antennae; CXE is widely expressed in larvae, with high expression genes in both male and female antennae; HyphSNMP1 and HyphSNMP2 are highly expressed in female and male antennae, respectively. This study laid a foundation for clarifying the molecular mechanism of sex pheromone communication in the American white moth, and also provided an important basis for exploring new ways of olfactory molecular control in the American white moth. The research has important reference significance and expands the molecular mechanism of olfactory in Lepidoptera moths.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S763.42

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