【摘要】：在大自然之中,低溫、氧化、高溫、土壤鹽堿化和干旱等都會(huì)對(duì)動(dòng)植物的生長(zhǎng)發(fā)育和生存產(chǎn)生不利的影響,例如長(zhǎng)勢(shì)弱小瘦弱,數(shù)量明顯減少,嚴(yán)重的還有可能造成絕種。根據(jù)以往的研究成果得知,轉(zhuǎn)錄因子和調(diào)節(jié)基因是介導(dǎo)動(dòng)植物響應(yīng)逆境脅迫的主要因素。轉(zhuǎn)錄輔激活因子跟轉(zhuǎn)錄因子的活性有著密切關(guān)系,目前對(duì)它的定義基本上都是,通過(guò)增強(qiáng)轉(zhuǎn)錄因子和順式作用元件(例如通用轉(zhuǎn)錄因子TBP)的結(jié)合,從而能夠達(dá)到增強(qiáng)轉(zhuǎn)錄活性的作用。核受體轉(zhuǎn)錄輔激活因子MBF1是一個(gè)在進(jìn)化上高度保守的序列,其基因序列從古細(xì)菌到人都是相當(dāng)保守的,并且它參與了內(nèi)皮細(xì)胞分化、激素調(diào)節(jié)、中樞神經(jīng)系統(tǒng)的發(fā)育、脂類(lèi)代謝和組氨酸新陳代謝等不同生物過(guò)程。MBF1首次被提純是從蠶的后部絲腺抽提物中,在不同生物體里的MBF1蛋白通過(guò)與GCN4、c-Jun、ATF1或其他核受體相互作用而與通用轉(zhuǎn)錄因子TBP連接,最后激活基因的轉(zhuǎn)錄。mbf1基因不僅是在進(jìn)化上高度保守,在功能上也是高度保守的,能參與到多種逆境脅迫反應(yīng)中,可以通過(guò)調(diào)節(jié)多種信號(hào)途徑以提高對(duì)不良環(huán)境的抵抗力。本研究以模式生物果蠅為研究材料,采用提取RNA,反轉(zhuǎn)錄,PCR的方法從野生型黑腹果蠅(Drosophila melanogaster)中克隆得到了全基因組的mbf1序列,其大小為4574bp,并將其命名為gmbf1。我們構(gòu)建了表達(dá)載體CaSpeR-gmbf1,通過(guò)顯微注射法,將CaSpeR-gmbf1質(zhì)粒轉(zhuǎn)入野生型果蠅中,獲得了P[gmbf1]轉(zhuǎn)基因果蠅。通過(guò)染色體置換方法,獲得了拯救品系P[gmbf1];mbf1-。分別對(duì)野生型、突變體、拯救品系和過(guò)表達(dá)品系的果蠅進(jìn)行冷脅迫處理,觀察其成活率情況,結(jié)果表明mbf1基因在冷脅迫中發(fā)揮了重要功能。并且在響應(yīng)冷脅迫過(guò)程中,通過(guò)熒光定量PCR實(shí)驗(yàn)發(fā)現(xiàn)mbf1基因的表達(dá)量升高。免疫熒光染色實(shí)驗(yàn)揭示了轉(zhuǎn)錄輔激活因子MBF1在低溫脅迫的過(guò)程里能夠從細(xì)胞質(zhì)中轉(zhuǎn)移到了細(xì)胞核中,進(jìn)一步說(shuō)明MBF1能夠在這一過(guò)程中發(fā)揮作用,從而實(shí)現(xiàn)調(diào)控目標(biāo)基因活性的目的,增強(qiáng)其抗寒性。利用Microarray實(shí)驗(yàn)鑒定出相比于野生型果蠅,mbf1突變體果蠅里有哪些基因的表達(dá)發(fā)生改變,從中選取與代謝相關(guān)的幾個(gè)基因,通過(guò)熒光定量PCR實(shí)驗(yàn)鑒定哪些基因與mbf1基因相關(guān),并且參與抗寒性。在mbf1突變體中,Nmdmc和CG5804的表達(dá)量降低,CG14893和CG11598的表達(dá)量升高。果蠅受到冷處理后,Nmdmc和CG5804的表達(dá)量升高,CG14893和CG11598的表達(dá)量降低。這些結(jié)果表明:MBF1可能通過(guò)調(diào)節(jié)這些與脂代謝相關(guān)的基因的表達(dá)來(lái)參與冷脅迫響應(yīng)。
[Abstract]:In nature, low temperature, oxidation, high temperature, soil salinization and drought will have adverse effects on the growth and survival of animals and plants. According to previous studies, transcription factors and regulatory genes are the main factors mediating the response of plants and animals to stress. Transcription coactivators are closely related to the activity of transcription factors. At present, they are basically defined by enhancing the binding of transcription factors to cis-acting elements, such as the universal transcription factor TBP. Thus, the transcriptional activity can be enhanced. Nuclear receptor transcriptional coactivator (MBF1) is an evolutionarily conserved sequence, and its gene sequence is quite conserved from archaea to human, and it is involved in endothelial cell differentiation, hormone regulation, and central nervous system development. Lipid metabolism and histidine metabolism are different biological processes. MBF1 was first purified from the posterior silk gland extract of silkworm, and the MBF1 protein in different organisms was purified through the interaction with GCN4,c-Jun,. ATF1 or other nuclear receptors interact with the common transcription factor TBP, and finally activate the transcription of the gene. The mbf1 gene is not only highly conserved in evolution, but also highly conserved in function, and can participate in many stress responses. Resistance to adverse environments can be improved by regulating multiple signal pathways. In this study, the model organism Drosophila melanogaster was used to extract RNA, reverse transcription.The whole genome mbf1 sequence was cloned from wild type Drosophila melanogaster (Drosophila melanogaster) by PCR method. Its size was 4574 BP, and it was named gmbf1.. We constructed the expression vector CaSpeR-gmbf1, and transformed the CaSpeR-gmbf1 plasmid into wild type Drosophila melanogaster by microinjection, and obtained P [gmbf1] transgenic Drosophila melanogaster. The rescue strain P [gmbf1]; mbf1-. was obtained by chromosome replacement method. Wild type, mutant, rescue strain and over-expressed strain were treated with cold stress, and the survival rate was observed. The results showed that mbf1 gene played an important role in cold stress. In response to cold stress, the expression of mbf1 gene was found to be increased by fluorescence quantitative PCR assay. Immunofluorescence staining revealed that transcriptional coactivator (MBF1) could be transferred from cytoplasm to nucleus during low temperature stress, which suggested that MBF1 could play a role in this process. In order to achieve the purpose of regulating the activity of the target gene, enhance its cold resistance. Compared with wild type Drosophila melanogaster, Microarray assay was used to identify which genes had changed in mbf1 mutant, and several genes related to metabolism were selected from them, and which genes were related to mbf1 gene were identified by fluorescence quantitative PCR experiment. And participate in cold resistance. In mbf1 mutant, the expression of Nmdmc and CG5804 decreased, and the expression of CG14893 and CG11598 increased. The expression of Nmdmc and CG5804 increased and the expression of CG14893 and CG11598 decreased after cold treatment. These results suggest that MBF1 may participate in cold stress response by regulating the expression of these genes related to lipid metabolism.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q78

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