中华蜜蜂气味受体基因Or57、Or139和Or167的克隆与时空表达分析

发布时间：2018-03-10 20:29

本文选题：中华蜜蜂　切入点：气味受体基因　出处：《山西农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：本研究以中华蜜蜂为研究对象,对其三个传统气味受体基因AcerOr57、AcerOrl39和AcerOrl67进行克隆、序列分析和定量表达分析,研究结果如下：1利用RT-PCR法获得了中华蜜蜂气味受体基因AcerOr57、AcerOrl39和AcerOr167的cDNA序列(GenBank登录号分别为：KT716393、KT239370和KT239369),包括完整的开放阅读框,分别编码407、387和436个氨基酸。生物信息学表明,这3个气味受体均为疏水性蛋白;分别具有6个、7个和5个跨膜结构域；均无信号肽和糖基化位点;AcerOr57和AcerOr139的结构不稳定,AcerOr167的结构较稳定;这与昆虫传统气味受体的结构特征相符合。2通过在NCBI数据库中Blastp搜索,分别找到与AcerOr57、AcerOrl39和AcerOr167的同源的序列,多序列比对发现AcerOr57与西方蜜蜂AmelOr57的相似性最高,为97%,且与AmelOr55、AmelOr56和AmelOr58均具有较高的相似性,推测AcerOr55、AcerOr56、AcerOr57和AcerOr58可能是蜜蜂在进化过程中通过基因复制产生的同源基因,且有可能串联排列在同一染色体上;AcerOr139也与西方蜜蜂的多个气味受体具有较高的相似性,其中与西方蜜蜂的假基因AmelOr139的相似性最高,为73%,对于AcerOr139是否也丧失原有功能,产生新的功能需要深入的研究；AcerOr167与膜翅目昆虫气味受体具有较高的的相似性,与西方蜜蜂AmelOr167的相似性最高,为94%,说明它们是直系同源基因。3利用实时荧光定量技术对AcerOr57, AcerOrl39和AcerOrl67在工蜂羽化后不同组织(触角、头、胸、腹、足、翅)和不同发育阶段(1、5、10、15、20、25和30日龄)中的相对表达量进行检测。结果表明,AcerOr57和AcerOr167 mRNA在工蜂各组织中均有表达,其中,在触角中的表达量极显著高于其他组织(P0.01); AcerOr139 mRNA也在工蜂各组织中均表达,但在不同的发育阶段表达特性不同,在1、5、25和30日龄时触角的表达量最高,10、15和20日龄时头部的表达量最高,推测这3个气味受体与蜜蜂的嗅觉识别作用有关。AcerOr57 mRNA在工蜂羽化后1日龄时表达量最高,10日龄最低,之后呈逐渐上升的趋势,推测其可能与中蜂出巢采集,识别花香气味有关;AcerOr139 mRNA在工蜂羽化后5、25和30日龄时均出现相对较高的表达,AcerOr167 mRNA在工蜂羽化后5日龄和20日龄时呈现相对较高的表达,推测这两个基因在中蜂识别花粉和花蜜气味分子过程中起着重要的作用。另外,AcerOr167 mRNA在工蜂整个发育阶段的表达量明显高于AcerOr57和AcerOr139,推测AcerOr167可能在中蜂的嗅觉系统中发挥着重要的作用
[Abstract]:In this study, the bees as the research object, the three traditional odorant receptor gene AcerOr57, AcerOrl39 and AcerOrl67 were cloned, expressed sequence analysis and quantitative analysis, the research results are as follows: 1 by using the RT-PCR method to obtain the bee odorant receptor gene AcerOr57, AcerOrl39 and AcerOr167 cDNA sequence (GenBank accession number: KT716393, KT239370 and KT239369), including the complete open reading frame encoding 407387 and 436 amino acids respectively. Bioinformatics showed that these 3 odorant receptors were hydrophobic protein; respectively with 6, 7 and 5 transmembrane domains; no signal peptide and glycosylation sites; AcerOr57 and AcerOr139 the instability of the structure of AcerOr167 is stable; and the traditional insect odorant receptor structure with.2 in the NCBI database by Blastp search, were found with AcerOr57, AcerOrl39 and AcerOr167 The homologous sequence, sequence comparison showed that the highest similarity, AcerOr57 and Western bees AmelOr57 was 97%, and the AmelOr55, AmelOr56 and AmelOr58 were similar, higher AcerOr55, AcerOr56, AcerOr57 and AcerOr58 may be the bees in the process of evolution by gene duplication homologous genes, and there may be arranged in series on the same chromosome; a plurality of odorant receptor AcerOr139 and Western bees have higher similarity, the highest similarity of pseudogene AmelOr139 with Western bees was 73%, for AcerOr139 is the loss of the original function, which further research need a new similarity function; AcerOr167 and odorant receptors with Hymenoptera the higher, and the highest, similar to Western bees AmelOr167 was 94%, indicating that they are orthologs of.3 using real-time fluorescence quantitative AcerOr57 technology of AcerOrl. 39 and AcerOrl67 in different tissues after emergence (worker antennae, head, chest, abdomen, feet, wings) and different developmental stages (1,5,10,15,20,25 and 30 days of age) relative expression quantity in the test. The results showed that AcerOr57 and AcerOr167 mRNA were expressed in various tissues in the expression of the worker, the antenna. Was significantly higher than that of other organizations (P0.01); AcerOr139 mRNA was also expressed in tissues of workers, but the expression in different developmental stages of different characteristics, expression of antenna in 1,5,25 and at the age of 30, the highest 10,15, and at the age of 20, the head of the highest expression, speculated that the 3 odor receptors and bees the role of.AcerOr57 in olfactory mRNA workers after emergence at the age of 1, the highest expression level at 10 days of age, the lowest, after gradually rising trend, suggesting that it may be with the bee nest collection, identification of floral scent; AcerOr139 mRNA and 30 5,25 after emergence in worker Relatively high expression showed age, AcerOr167 mRNA in worker 5 after emergence and 20 days of age showed a relatively higher level, it is suggested that the two gene plays an important role in the identification of bee pollen and nectar odor molecules in the process. In addition, the AcerOr167 expression of mRNA in the whole growth stage of worker the amount was significantly higher than that of AcerOr57 and AcerOr139, AcerOr167 speculated that the olfactory system may in the bee play an important role in

【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S891;Q78

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