本文選題：蜜蜂 + 勞動(dòng)分工��； 參考：《福建農(nóng)林大學(xué)》2017年碩士論文

【摘要】：Takeout(to)基因是一種節(jié)律調(diào)控輸出基因,廣泛存在于昆蟲的營(yíng)養(yǎng)功能和化學(xué)感受等相關(guān)組織。to基因主要參與昆蟲的生長(zhǎng)發(fā)育、行為調(diào)節(jié)及多種生理代謝過程。為探究to基因在蜜蜂中的表達(dá)規(guī)律和相關(guān)生物學(xué)功能,我們克隆了中華蜜蜂Apis cerana cerana和意大利蜜蜂Apis mellifera ligustica的to基因,分析該基因在哺育蜂和采集蜂各組織的mRNA表達(dá)規(guī)律,并利用RNA干擾技術(shù)進(jìn)一步探討了其生物學(xué)功能。主要研究結(jié)果如下:通過反轉(zhuǎn)錄多聚酶鏈?zhǔn)椒磻?yīng)技術(shù)(RT-PCR)首次克隆獲得了中華蜜蜂和意大利蜜蜂to基因的cDNA序列分別為1058 bp和783 bp,依次命名為AcTO1和AmTO1,其開放閱讀框(ORF)長(zhǎng)度均為738 bp,均編碼246個(gè)氨基酸,預(yù)測(cè)的蛋白質(zhì)分子質(zhì)量分別為27.09和27.07 kDa,等電點(diǎn)pI均為8.40。生物信息學(xué)分析結(jié)果表明,AcTO1和AmTO1蛋白在N端均有一段信號(hào)肽序列,無跨膜結(jié)構(gòu),均為分泌蛋白。此外,2種蛋白的氨基酸序列中都存在一個(gè)保幼激素結(jié)合蛋白(JHBP)超家族的保守結(jié)構(gòu)域。采用熒光定量PCR技術(shù)qRT-PCR對(duì)中華蜜蜂和意大利蜜蜂的3種采集蜂(正常年齡采集蜂1和2、提前采集蜂)和2種哺育蜂(正常年齡哺育蜂和超齡哺育蜂)在頭部、胸部和腹部這3種組織對(duì)應(yīng)的的to基因表達(dá)量進(jìn)行檢測(cè)和分析。結(jié)果表明,to基因在3種采集蜂和2種哺育蜂的各組織均有表達(dá),相對(duì)表達(dá)量從高到低依次為:頭部、胸部、腹部。重要的是to基因在中華蜜蜂和意大利蜜蜂的表達(dá)模式都具有組織依賴型,主要在頭部表達(dá),并且所有采集蜂頭部表達(dá)量都高于所有的哺育蜂,說明該基因可能參與調(diào)控蜜蜂的勞動(dòng)分工。為深入探究AmTO1基因是否參與調(diào)控蜜蜂的勞動(dòng)分工,我們選取16日齡意大利蜜蜂工蜂為試驗(yàn)材料,并分別飼喂AmTO1 dsRNA,DEPC H2O(空白對(duì)照)和DsRed(陰性對(duì)照)。結(jié)果表明,干擾組(AmTO1 dsRNA)蜜蜂頭部AmTO1基因的表達(dá)量顯著低于對(duì)照組(DEPC H2O和DsRed),并且工蜂的出巢率明顯低于對(duì)照組。以上結(jié)果表明:to基因在蜜蜂的采集行為方面可能發(fā)揮著重要功能。
[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.
【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S891

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