中华蜜蜂和意大利蜜蜂takeout基因的克

发布时间：2018-04-16 03:08

本文选题：蜜蜂 + 劳动分工　；参考：《福建农林大学》2017年硕士论文

【摘要】：Takeout(to)基因是一种节律调控输出基因,广泛存在于昆虫的营养功能和化学感受等相关组织。to基因主要参与昆虫的生长发育、行为调节及多种生理代谢过程。为探究to基因在蜜蜂中的表达规律和相关生物学功能,我们克隆了中华蜜蜂Apis cerana cerana和意大利蜜蜂Apis mellifera ligustica的to基因,分析该基因在哺育蜂和采集蜂各组织的mRNA表达规律,并利用RNA干扰技术进一步探讨了其生物学功能。主要研究结果如下:通过反转录多聚酶链式反应技术(RT-PCR)首次克隆获得了中华蜜蜂和意大利蜜蜂to基因的cDNA序列分别为1058 bp和783 bp,依次命名为AcTO1和AmTO1,其开放阅读框(ORF)长度均为738 bp,均编码246个氨基酸,预测的蛋白质分子质量分别为27.09和27.07 kDa,等电点pI均为8.40。生物信息学分析结果表明,AcTO1和AmTO1蛋白在N端均有一段信号肽序列,无跨膜结构,均为分泌蛋白。此外,2种蛋白的氨基酸序列中都存在一个保幼激素结合蛋白(JHBP)超家族的保守结构域。采用荧光定量PCR技术qRT-PCR对中华蜜蜂和意大利蜜蜂的3种采集蜂(正常年龄采集蜂1和2、提前采集蜂)和2种哺育蜂(正常年龄哺育蜂和超龄哺育蜂)在头部、胸部和腹部这3种组织对应的的to基因表达量进行检测和分析。结果表明,to基因在3种采集蜂和2种哺育蜂的各组织均有表达,相对表达量从高到低依次为:头部、胸部、腹部。重要的是to基因在中华蜜蜂和意大利蜜蜂的表达模式都具有组织依赖型,主要在头部表达,并且所有采集蜂头部表达量都高于所有的哺育蜂,说明该基因可能参与调控蜜蜂的劳动分工。为深入探究AmTO1基因是否参与调控蜜蜂的劳动分工,我们选取16日龄意大利蜜蜂工蜂为试验材料,并分别饲喂AmTO1 dsRNA,DEPC H2O(空白对照)和DsRed(阴性对照)。结果表明,干扰组(AmTO1 dsRNA)蜜蜂头部AmTO1基因的表达量显著低于对照组(DEPC H2O和DsRed),并且工蜂的出巢率明显低于对照组。以上结果表明:to基因在蜜蜂的采集行为方面可能发挥着重要功能。
[Abstract]:Takeouttotogene is a rhythmically regulated exportation gene, which is widely found in insect related tissues, such as nutritional function and chemical sensitivity, mainly involved in insect growth and development, behavioral regulation and a variety of physiological processes of metabolism.In order to investigate the expression of to gene in honeybees and its related biological functions, we cloned the to genes of Apis cerana cerana and Apis mellifera ligustica of Apis mellifera chinensis, and analyzed the mRNA expression patterns of these genes in the tissues of nursing bees and collecting bees.The biological function was further discussed by using RNA interference technique.The main results are as follows: by reverse transcriptase polymerase chain reaction (RT PCR), the cDNA sequence of bee to gene was 1058 BP and 783bprespectively, named AcTO1 and AmTO1, respectively, and its open reading frame was obtained.The length of ORF is 738 BP and encodes 246 amino acids.The predicted molecular weight of protein is 27.09 and 27.07 kDa, and the isoelectric point Pi is 8.40.The results of bioinformatics analysis showed that both AcTO1 and AmTO1 proteins had a sequence of signal peptides at the N-terminal, which had no transmembrane structure and were secreted proteins.In addition, there is a conserved domain of JHBP superfamily in the amino acid sequences of the two proteins.Fluorescence quantitative PCR (PCR) technique was used to detect three species of bees (1 and 2 from normal age) and two species of nursing bees (normal age and over age) from honeybee and Italian bee.The expression of to gene in thoracic and abdominal tissues was detected and analyzed.The results showed that the gene was expressed in all tissues of three collected bees and two kinds of nursing bees, and the relative expression was in the order of head, chest and abdomen from high to low.The important thing is that the expression patterns of to gene in Chinese bee and Italian bee are tissue dependent, mainly expressed in the head, and the expression of to gene in the head of all collected bees is higher than that in all nursing bees.It suggests that the gene may be involved in regulating the division of labor in honeybees.In order to investigate whether the AmTO1 gene is involved in the regulation of labor division of honeybees, 16 day old worker bees of Italian bees were selected as experimental materials and fed AmTO1 dsRNA-DEPC H _ 2O (blank control) and DsRed (negative control), respectively.The results showed that the expression of AmTO1 gene in the head of the interference group was significantly lower than that in the control group, and the nesting rate of the worker bee was significantly lower than that of the control group.These results suggest that the 1 to gene may play an important role in the behavior of bees.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S891

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