本文選題：油菜 + 擬南芥��； 參考：《西北農林科技大學》2017年碩士論文

【摘要】：MYB(myeloblastosis)轉錄因子家族作為植物中最大的轉錄因子家族之一,在植物的生長發(fā)育過程中發(fā)揮著重要的作用。它們還調節(jié)次級代謝過程,例如苯丙烷類化合物的代謝;并調控對于多種生物和非生物逆境的響應過程。同時,在不同的調節(jié)過程中,又有多種激素等信號通路參與,形成了復雜的的調控網絡。雖然,有關模式植物擬南芥中MYB的功能研究的報道頗多,但仍有很多MYB基因的功能未知。并且,與擬南芥同屬十字花科的甘藍型油菜(Brassica napus L.)的MYB基因的報道非常少。因此,有必要開展研究。在本研究中,我們在前期有關油菜R3R3-MYB轉錄因子基因的鑒定、cDNA克隆、轉錄活性檢測、亞細胞定位分析以及功能篩選的基礎上,較為系統地研究了BnaMYB78基因的功能與分子調控機制。BnaMYB78在煙草中瞬時的過表達會導致活性氧(reactive oxygen species,ROS)的積累并誘發(fā)細胞死亡,并通過隨后的二氨基聯苯胺(3,3’-diaminobenzidine,DAB)染色、葉綠素、丙二醛、過氧化氫等的含量測定等一系列生理指標的檢測證實了這一現象。實時熒光定量RT-PCR(quantitative real-time RT-PCR,qRT-PCR)分析顯示BnaMYB78在油菜衰老葉片里表達水平偏高。BnaMYB78是一個轉錄激活蛋白,定位于細胞核內。并且,實時熒光定量RT-PCR篩查發(fā)現,BnaMYB78的下游靶標基因可能包括RbohB、PR2、PR5、GST、ACRE31、HIN1等,并進一步通過雙熒光素酶報告(dual-luciferase,dual-LUC)基因系統,發(fā)現BnaMYB78很可能與三種不同的MYB順式作用元件結合,并且驗證了BnaMYB78對GST和HIN1具有轉錄調控作用。我們在試驗過程中發(fā)現,擬南芥(Arabidopsis thaliana)中R2R3-MYB轉錄因子家族成員AtMYBx的過表達株系在黑暗誘導下幼苗衰老會提前,且正常生長條件下葉片衰老也會提前。通過實時熒光定量RT-PCR分析顯示AtMYBx的表達被JA顯著誘導。AtMYBx是一個轉錄激活蛋白,定位于細胞核內。雙熒光素酶報告基因系統結合電遷移變動分析(electrophoretic mobility shift assay,EMSA)試驗表明,AtMYBx能結合三種不同的MYB順式作用元件。然后,檢測了茉莉酸和衰老相關的標志基因在AtMYBx的突變體與過表達株系中的轉錄本水平,發(fā)現部分茉莉酸合成和信號轉導相關的標志基因的轉錄本水平在AtMYBx的過表達株系中發(fā)生了顯著地變化。并進一步通過雙熒光素酶報告基因系統,驗證了AtMYBx對部分基因的轉錄調控作用。同時,又通過激素的定量測定,發(fā)現AtMYBx過表達會導致JA積累增加,說明AtMYBx能夠正調控LOX2等茉莉酸合成相關基因,使植物體內茉莉酸合成和積累增加,從而誘發(fā)植物提前衰老。本研究不僅發(fā)現了兩個新的分別調控活性氧與茉莉酸信號轉導的R2R3-MYB型基因,明確了它們的表達與轉錄活性等特性,而且對下游靶標基因與分子調控機制進行了鑒定與初步解析,為深入地認識R2R3-MYB轉錄因子基因的生物學功能和調控機理奠定基礎。
[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.
【學位授予單位】：西北農林科技大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2

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本文編號：1991886