油菜与拟南芥中两个MYB转录因子基因分别调控活性氧与茉莉酸信号转导的分子机制研究

发布时间：2018-06-07 16:50

本文选题：油菜 + 拟南芥　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：MYB(myeloblastosis)转录因子家族作为植物中最大的转录因子家族之一,在植物的生长发育过程中发挥着重要的作用。它们还调节次级代谢过程,例如苯丙烷类化合物的代谢;并调控对于多种生物和非生物逆境的响应过程。同时,在不同的调节过程中,又有多种激素等信号通路参与,形成了复杂的的调控网络。虽然,有关模式植物拟南芥中MYB的功能研究的报道颇多,但仍有很多MYB基因的功能未知。并且,与拟南芥同属十字花科的甘蓝型油菜(Brassica napus L.)的MYB基因的报道非常少。因此,有必要开展研究。在本研究中,我们在前期有关油菜R3R3-MYB转录因子基因的鉴定、cDNA克隆、转录活性检测、亚细胞定位分析以及功能筛选的基础上,较为系统地研究了BnaMYB78基因的功能与分子调控机制。BnaMYB78在烟草中瞬时的过表达会导致活性氧(reactive oxygen species,ROS)的积累并诱发细胞死亡,并通过随后的二氨基联苯胺(3,3’-diaminobenzidine,DAB)染色、叶绿素、丙二醛、过氧化氢等的含量测定等一系列生理指标的检测证实了这一现象。实时荧光定量RT-PCR(quantitative real-time RT-PCR,qRT-PCR)分析显示BnaMYB78在油菜衰老叶片里表达水平偏高。BnaMYB78是一个转录激活蛋白,定位于细胞核内。并且,实时荧光定量RT-PCR筛查发现,BnaMYB78的下游靶标基因可能包括RbohB、PR2、PR5、GST、ACRE31、HIN1等,并进一步通过双荧光素酶报告(dual-luciferase,dual-LUC)基因系统,发现BnaMYB78很可能与三种不同的MYB顺式作用元件结合,并且验证了BnaMYB78对GST和HIN1具有转录调控作用。我们在试验过程中发现,拟南芥(Arabidopsis thaliana)中R2R3-MYB转录因子家族成员AtMYBx的过表达株系在黑暗诱导下幼苗衰老会提前,且正常生长条件下叶片衰老也会提前。通过实时荧光定量RT-PCR分析显示AtMYBx的表达被JA显著诱导。AtMYBx是一个转录激活蛋白,定位于细胞核内。双荧光素酶报告基因系统结合电迁移变动分析(electrophoretic mobility shift assay,EMSA)试验表明,AtMYBx能结合三种不同的MYB顺式作用元件。然后,检测了茉莉酸和衰老相关的标志基因在AtMYBx的突变体与过表达株系中的转录本水平,发现部分茉莉酸合成和信号转导相关的标志基因的转录本水平在AtMYBx的过表达株系中发生了显著地变化。并进一步通过双荧光素酶报告基因系统,验证了AtMYBx对部分基因的转录调控作用。同时,又通过激素的定量测定,发现AtMYBx过表达会导致JA积累增加,说明AtMYBx能够正调控LOX2等茉莉酸合成相关基因,使植物体内茉莉酸合成和积累增加,从而诱发植物提前衰老。本研究不仅发现了两个新的分别调控活性氧与茉莉酸信号转导的R2R3-MYB型基因,明确了它们的表达与转录活性等特性,而且对下游靶标基因与分子调控机制进行了鉴定与初步解析,为深入地认识R2R3-MYB转录因子基因的生物学功能和调控机理奠定基础。
[Abstract]:MYB myeloblastosis) transcription factor family, as one of the largest transcription factor families in plants, plays an important role in the growth and development of plants. They also regulate secondary metabolic processes, such as the metabolism of phenylpropane compounds, and the response to multiple biological and abiotic stresses. At the same time, in the different regulation process, there are a variety of hormones and other signal pathways involved, forming a complex regulatory network. Although there are many reports about the function of MYB in Arabidopsis thaliana, there are still many unknown functions of MYB gene. In addition, Brassica napus L., which belongs to the same family as Arabidopsis thaliana, belongs to Cruciferae. Very few reports of MYB genes have been reported. Therefore, it is necessary to carry out research. In this study, we based on the previous identification of R3R3-MYB transcription factor gene in rapeseed, the detection of transcriptional activity, subcellular localization and functional screening. The function and molecular regulatory mechanism of BnaMYB78 gene. BnaMYB78 in tobacco were systematically studied. The transient overexpression of BnaMYB78 resulted in the accumulation of reactive oxygen species-ROSs (Ros) and induced cell death. The determination of malondialdehyde, hydrogen peroxide and a series of physiological indexes confirmed this phenomenon. Real-time fluorescence quantitative RT-PCR(quantitative real-time RT-PCRQRT-PCRanalysis showed that the high expression level of BnaMYB78 in senescent leaves of rape showed that BnaMYB78 was a transcriptional activator and located in the nucleus. Moreover, real-time fluorescence quantitative RT-PCR screening revealed that the downstream target gene of BnaMYB78 might include RbohBHBP2PR2 / PR5 GSTACRE31HIN1, etc. Furthermore, by using the dual-luciferase report system, we found that BnaMYB78 probably binds to three different MYB cis-acting elements. The transcriptional regulation of GST and HIN1 by BnaMYB78 was verified. We found that the over-expression of AtMYBx, a member of the R2R3-MYB transcription factor family, in Arabidopsis thaliana in Arabidopsis, could lead to early senescence in seedlings induced by darkness and early senescence in leaves under normal growth conditions. Real-time fluorescence quantitative RT-PCR analysis showed that the expression of AtMYBx was significantly induced by JA. AtMYBx was a transcriptional activator and located in the nucleus. The double luciferase reporter gene system combined with electromigration variation analysis showed that AtMYBx could bind to three different MYB cis-acting elements. Then, we detected the transcription level of jasmonic acid and senescence related marker genes in AtMYBx mutants and overexpression lines. It was found that the transcription level of some marker genes related to jasmonic acid synthesis and signal transduction changed significantly in AtMYBx overexpression lines. Furthermore, the transcriptional regulation of some genes by AtMYBx was verified by double luciferase reporter gene system. At the same time, through the quantitative determination of hormones, it was found that the overexpression of AtMYBx would lead to the increase of JA accumulation, which indicated that AtMYBx could regulate the synthesis and accumulation of jasmonic acid related genes such as LOX2, increase the synthesis and accumulation of jasmonic acid in plants, and induce plant senescence ahead of time. In this study, not only two new R2R3-MYB type genes were found to regulate the signal transduction of reactive oxygen species and jasmonic acid, but also their expression and transcriptional activities were clarified, and the downstream target genes and molecular regulatory mechanisms were identified and analyzed. It lays a foundation for further understanding the biological function and regulation mechanism of R2R3-MYB transcription factor gene.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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