蜜蜂幼虫饥饿信息素鉴定及其发育机理研究

发布时间：2018-04-19 08:29

  本文选题：蜜蜂 + 幼虫　； 参考：《江西农业大学》2016年博士论文

【摘要】：蜜蜂是一种典型的真社会性昆虫,它的独特生物学特性一直受广大科学研究者的关注。本研究以意大利蜜蜂(Apis mellifera ligustica,简称意蜂)为实验材料,利用Needle trap技术与气质联用技术分析鉴定出9种工蜂幼虫信息素。发现E-β-罗勒烯为工蜂幼虫的饥饿信息素,在饥饿组幼虫中含量显著高于饲喂组及纯食物组,且2日龄幼虫组显著高于4日龄幼虫组。同时,行为学实验结果表明添加E-β-罗勒烯可引起哺育蜂的探头哺育行为,进一步证实了E-β-罗勒烯为工蜂幼虫饥饿信息素。利用RNA-Seq技术分析鉴定出了E-β-罗勒烯在幼虫体内的生物合成通路,并且发现参与该通路的3个基因在2d幼虫中表达显著高于4d幼虫,并在饥饿30分钟时表达量达到最高峰。利用上述相同的Needle trap与气质联用技术,分析鉴定出了蜂王幼虫与雄蜂幼虫的饥饿信息素。实验结果表明,蜂王与雄蜂幼虫也以E-β-罗勒烯作为其饥饿信息素。各饥饿幼虫组所含E-β-罗勒烯均显著高于饲喂幼虫组与食物组。2日龄蜂王、雄蜂与工蜂幼虫饥饿组E-β-罗勒烯含量均差异不显著,但4日龄蜂王幼虫饥饿组E-β-罗勒烯含量显著低于雄蜂与工蜂幼虫组。同时,发现蜂王幼虫含有一种雄蜂与工蜂幼虫没有的幼虫信息素----2-庚酮。RNA-Seq结果表明,蜂王与雄蜂幼虫也具有与工蜂幼虫相同的E-β-罗勒烯生物合成通路与相关基因。使用RNA-Seq测序技术检测了意蜂三型蜂2日龄与4日龄幼虫的mRNA,分析比较了三型蜂幼虫在发育过程中的基因表达差异。各三型蜂幼虫组检测到的总基因个数没有差异,但各组之间存在大量差异表达基因。2日龄雄蜂幼虫与蜂王幼虫对比组差异表达基因数为475个,高于雄蜂与工蜂幼虫对比组(197个)及蜂王与工蜂幼虫对比组(121个);4日龄蜂王与雄蜂幼虫对比组差异基因达687个,工蜂与雄蜂幼虫对比组为604个,而蜂王与工蜂幼虫对比组仅为475个,表明雄蜂幼虫与其他两种雌性蜂之间的差异高于两种雌性蜂之间的差异。而4日龄各对比组的差异基因数明显高于2日龄对比组,表明随着日龄的增加三型蜂幼虫的发育更为分化。其次,2d与4d工蜂幼虫之间差异表达基因为1190个,2d与4d蜂王幼虫对比组为1181个,而雄蜂幼虫对比组仅为598个,表明蜜蜂单双倍体遗传背景在其发育上的调控模式存在差异。基因共表达聚类分析结果表明2日龄工蜂与雄蜂幼虫先聚类在一起,再与2日龄蜂王聚类,4日龄则两种雌性蜂聚类更近。雌性蜜蜂幼虫与雄蜂幼虫相比较,发现大量差异表达基因参与了蜜蜂激素合成分泌、蜂毒的合成、眼睛和飞行肌肉的生长发育、眼睛发育、卵巢成熟、以及一些调控基因表达的转录因子与信号通路等,这与雌性蜂与雄蜂在形态、生理与行为上的特征相符。
[Abstract]:Honeybee is a typical true-social insect, its unique biological characteristics have been concerned by many scientific researchers.In this study, 9 species of pheromones of worker bee larvae were identified by means of Needle trap and GC-MS using Apis mellifera ligustica (Italian bee) as the experimental material.It was found that E- 尾 -basilerene was the pheromone of worker bee larvae, and the content of E- 尾 -basilerene in the starved larvae was significantly higher than that in the feeding and pure food groups, and that in the 2-day-old larvae was significantly higher than that in the 4-day-old larvae.At the same time, the results of behavioral experiments showed that the addition of E- 尾 -basilerene could induce the feeding behavior of nursing bees, which further confirmed that E- 尾 -basilerene was a hunger pheromone of worker bee larvae.The biosynthesis pathway of E- 尾 -basilerene in larva was identified by RNA-Seq technique. It was found that the three genes involved in the biosynthesis pathway were significantly higher in 2 d larva than in 4 d larva, and reached the highest level at 30 minutes of starvation.By using the same Needle trap and GC-MS techniques, the hunger pheromones of the queen bee larvae and the male larvae were analyzed and identified.The results showed that E- 尾-basilerene was also used as the hunger pheromone of the queen bee and the larvae of the male bee.The content of E- 尾-basilerene in the starved larva group was significantly higher than that in the feeding larval group and the feeding group. The content of E- 尾-basilerene in the starved group of male bee and worker bee was not significantly different from that in the feeding group and the food group.However, the content of E- 尾-basilerene in the starvation group was significantly lower than that in the male and worker larvae groups.At the same time, it was found that the queen bee larva contained a pheromone 2-heptanone, RNA-Seq, which was not found in the male bee and worker bee larvae. The results showed that the queen bee larva and the male bee larva also had the same E- 尾 -basilerene biosynthesis pathway and related genes as the worker bee larvae.The RNA-Seq sequencing technique was used to detect the mRNAs of the 2-day-old and 4-day-old larvae of the Italian bee, and the difference of gene expression during the development of the larvae was analyzed and compared.There was no difference in the number of total genes detected in each larvae group, but there were a large number of differentially expressed genes between the groups. The number of differentially expressed genes between the two groups was 475.There were 687 differentially expressed genes in the control group (197) higher than those in the control group (197) and the control group (121) and 604 (P < 0.05) respectively, and the difference was significantly higher than that in the control group (n = 197) and the control group (n = 121) and the control group (n = 604) respectively, which were higher than those in the control group (n = 197).The difference between male bee larva and other two female wasps was higher than that between two female wasps, but the comparison group between queen bee and worker bee larvae was 475, which indicated that the difference between male bee larva and other two kinds of female wasps was higher than that between them.The number of differentially expressed genes in the 4-day-old control group was significantly higher than that in the 2-day-old control group, indicating that the development of the larvae of the three-type wasp was more differentiated with the increase of the age of 4 days.Secondly, there were different expression bases between worker bee larvae (2d and 4d), because 1181 were in the contrast group of 1190 larva and 1181 in the control group, and only 598 in the control group, which indicated that there were differences in the developmental regulation pattern of the haploidy genetic background of honeybee.The results of gene coexpression cluster analysis showed that the two species of female wasps were clustered closer to the 2-day-old worker bee and the male bee larva, and then to the 2-day-old bee king cluster at 4 days old.A large number of differentially expressed genes were found to be involved in the synthesis and secretion of bee hormones, the synthesis of bee venom, the growth and development of eyes and flying muscles, the development of eyes, and the maturation of ovaries.Some transcriptional factors and signaling pathways regulating gene expression were found to be consistent with the morphological, physiological and behavioral characteristics of female and male bees.
【学位授予单位】：江西农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S891
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本文编号：1772342