果蝇MBF1通过调控代谢相关基因表达响应冷胁迫
发布时间：2019-01-07 06:49
【摘要】：在大自然之中,低温、氧化、高温、土壤盐碱化和干旱等都会对动植物的生长发育和生存产生不利的影响,例如长势弱小瘦弱,数量明显减少,严重的还有可能造成绝种。根据以往的研究成果得知,转录因子和调节基因是介导动植物响应逆境胁迫的主要因素。转录辅激活因子跟转录因子的活性有着密切关系,目前对它的定义基本上都是,通过增强转录因子和顺式作用元件(例如通用转录因子TBP)的结合,从而能够达到增强转录活性的作用。核受体转录辅激活因子MBF1是一个在进化上高度保守的序列,其基因序列从古细菌到人都是相当保守的,并且它参与了内皮细胞分化、激素调节、中枢神经系统的发育、脂类代谢和组氨酸新陈代谢等不同生物过程。MBF1首次被提纯是从蚕的后部丝腺抽提物中,在不同生物体里的MBF1蛋白通过与GCN4、c-Jun、ATF1或其他核受体相互作用而与通用转录因子TBP连接,最后激活基因的转录。mbf1基因不仅是在进化上高度保守,在功能上也是高度保守的,能参与到多种逆境胁迫反应中,可以通过调节多种信号途径以提高对不良环境的抵抗力。本研究以模式生物果蝇为研究材料,采用提取RNA,反转录,PCR的方法从野生型黑腹果蝇(Drosophila melanogaster)中克隆得到了全基因组的mbf1序列,其大小为4574bp,并将其命名为gmbf1。我们构建了表达载体CaSpeR-gmbf1,通过显微注射法,将CaSpeR-gmbf1质粒转入野生型果蝇中,获得了P[gmbf1]转基因果蝇。通过染色体置换方法,获得了拯救品系P[gmbf1];mbf1-。分别对野生型、突变体、拯救品系和过表达品系的果蝇进行冷胁迫处理,观察其成活率情况,结果表明mbf1基因在冷胁迫中发挥了重要功能。并且在响应冷胁迫过程中,通过荧光定量PCR实验发现mbf1基因的表达量升高。免疫荧光染色实验揭示了转录辅激活因子MBF1在低温胁迫的过程里能够从细胞质中转移到了细胞核中,进一步说明MBF1能够在这一过程中发挥作用,从而实现调控目标基因活性的目的,增强其抗寒性。利用Microarray实验鉴定出相比于野生型果蝇,mbf1突变体果蝇里有哪些基因的表达发生改变,从中选取与代谢相关的几个基因,通过荧光定量PCR实验鉴定哪些基因与mbf1基因相关,并且参与抗寒性。在mbf1突变体中,Nmdmc和CG5804的表达量降低,CG14893和CG11598的表达量升高。果蝇受到冷处理后,Nmdmc和CG5804的表达量升高,CG14893和CG11598的表达量降低。这些结果表明:MBF1可能通过调节这些与脂代谢相关的基因的表达来参与冷胁迫响应。
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78
本文编号：2403312
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q78
【参考文献】
相关期刊论文 前5条
1 赵春梅;张文彦;刘箭;;过量表达内质网小分子热激蛋白增强转基因番茄果实的耐冷藏性[J];西北植物学报;2011年01期
2 张毅;张岗;董艳玲;郭军;黄丽丽;康振生;;条锈菌诱导的小麦MBF1转录辅激活因子基因的克隆及其特征分析[J];作物学报;2009年01期
3 杨金莹;孙颖;孙爱清;伊淑莹;刘箭;;番茄LeHsp110/ClpB基因的分子克隆及其对植物耐热性的影响[J];生物工程学报;2006年01期
4 王丽,赵春梅,王义菊,刘箭;过量表达叶绿体小分子热激蛋白提高番茄的抗寒性(英文)[J];植物生理与分子生物学学报;2005年02期
5 刘强,张贵友,陈受宜;植物转录因子的结构与调控作用[J];科学通报;2000年14期
，本文编号：2403312
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2403312.html
最近更新
教材专著
