斜纹夜蛾性信息素合成途径相关基因的克隆和研究

发布时间：2018-03-09 09:45

本文选题：斜纹夜蛾　切入点：性信息素　出处：《中国计量学院》2015年硕士论文　论文类型：学位论文

【摘要】：斜纹夜蛾(Spodoptera litura)是危害性严重的鳞翅目害虫,它具有进化完善的性信息素通讯系统。斜纹夜蛾雌虫在信息素腺体释放性信息素,并通过它来吸引雄虫进而引发后续的交配行为。斜纹夜蛾性信息素是4种14碳直链不饱和脂肪酸酯类衍生物的混合物,它们的合成经历了软脂酸合成、碳链缩短反应、脱饱和作用和官能基团的修饰这一系列过程。在该途径中,多种酶参与其中并催化不同的生化反应。本研究参照已有的斜纹夜蛾转录组数据并结合斜纹夜蛾的性信息素合成途径,我们对其中的一系列相关基因进行了筛选,并将之与NCBI数据库中其他物种的性信息素合成途径基因进行比对,最终筛选到包括脂肪酸合成酶系、β氧化酶系、脱饱和酶、脂肪酰辅酶A还原酶和乙酰辅酶A转移酶5大类共90个基因。为了进一步确认它们的表达特征,我们分别以雌、雄斜纹夜蛾为模板通过PCR进行了半定量筛选。结果显示,大多数基因在雌、雄斜纹夜蛾腹部表达没有显著差异;然而,我们发现少部分基因在雌性个体中特异性表达;另外还有一部分基因在雌雄个体中的表达水平有显著差异,因此我们通过荧光定量PCR进一步检测它们的表达水平并计算出表达差异的倍数,结果表明这部分基因在雌虫中的表达水平均高于雄虫3-10倍;我们针对雌虫特异性表达基因,研究了它们在雌虫信息素腺体和腹部其他组织中的表达,结果表明这些基因绝大多数在信息素腺体中的表达水平显著高于腹部其他组织(p0.05),只有极少数基因在腹部其他组织中的表达量高于性信息素腺体。此外,我们选择了上述筛选到的属于不同类型(脂肪酸合成酶、脱饱和酶、脂酰辅酶A还原酶),且具有不同的表达特征的9个基因作进一步的研究。克隆了这9个基因并合成了双链RNA,然后将双链RNA注射到斜纹夜蛾雌虫进行基因沉默,通过荧光定量PCR检测基因沉默效果,根据GC-MS分析基因沉默后的雌虫性信息素组分的变化。结果表明注射双链RNA后对应的基因表达量降低,并且对Des3,Des5和FAR17沉默后发现雌虫产生的4中性信息素组分相对比例发生了变化。最后我们研究了雌虫特异性基因在不同地理区系的表达差异,结果表明大多数基因在不同地域表达有显著差异,而有的基因在不同地域表达水平相对稳定。研究斜纹夜蛾性信息素合成途径相关基因的功能和不同地理区系表达特征,有助于我们理解性信息素的种内变异并推测它的进化方向,此外为生物合成性信息扰乱蛾类交配提供理论依据,也为在田间应用性信息素防治害虫提供了重要的支持。
[Abstract]:Spodoptera litura (Spodoptera litura) is a serious pest of Lepidoptera, which has a well-evolved sex pheromone communication system. Females of Spodoptera litura release sex pheromone in pheromone glands. The sex pheromone of Spodoptera litura is a mixture of four 14 carbon straight chain unsaturated fatty acid esters, which undergo palmitic acid synthesis and carbon chain shortening. Desaturation and modification of functional groups. In this pathway, According to the transcriptional data of Spodoptera litura and the sex pheromone synthesis pathway of Spodoptera litura, a series of related genes were screened. The genes of sex pheromone biosynthesis pathway of other species in NCBI database were compared, and finally selected to include fatty acid synthase, 尾 oxidase, desaturase. There are 90 genes in 5 categories of fatty acyl coenzyme A reductase and acetyl coenzyme A transferase. In order to further confirm their expression characteristics, we used female and male Spodoptera litura as templates to carry out semi-quantitative screening by PCR. There was no significant difference in the abdominal expression of most genes between female and male Spodoptera litura, however, we found that a few genes were specifically expressed in female individuals, and there were also significant differences in the expression levels of some genes in female and male individuals. Therefore, we further detected their expression level by fluorescence quantitative PCR and calculated the multiple of the difference in expression. The results showed that the expression level of these genes in females was 3-10 times higher than that in males, and we expressed genes specifically for females. Having studied their expression in female pheromone glands and other abdominal tissues, The results showed that the expression level of these genes was significantly higher in pheromone glands than in other abdominal tissues, and only a few genes expressed more in other abdominal tissues than in sex pheromones. We selected different types of enzymes (fatty acid synthase, desaturase, desaturase). Nine genes with different expression characteristics were further studied. The nine genes were cloned and synthesized double-stranded RNAs. Then double-stranded RNA was injected into female Spodoptera litura for gene silencing. The effect of gene silencing was detected by fluorescence quantitative PCR, and the changes of sex pheromone components of female worm after gene silencing were analyzed according to GC-MS. The results showed that the corresponding gene expression decreased after injection of double-stranded RNA. The relative proportion of 4 neutral pheromones produced by female worms was changed after the silencing of Des3, Des5 and FAR17. Finally, we studied the difference of female specific gene expression in different geographical regions. The results showed that most genes expressed differently in different regions, while some genes were relatively stable in different regions. The function of genes related to sex pheromone biosynthesis pathway of Spodoptera litura and the expression characteristics of different geographical regions were studied. It is helpful for us to understand the intraspecific variation of sex pheromone and to speculate its evolutionary direction. In addition, it provides a theoretical basis for biosynthetic information to disrupt the mating of moths, and also provides important support for the application of pheromones to pest control in the field.
【学位授予单位】：中国计量学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4

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