發(fā)布時間：2018-06-06 10:38

  本文選題：油菜素甾醇 + 油菜素內(nèi)酯　； 參考：《蘭州大學》2017年博士論文

【摘要】：油菜素甾醇(brassinosteroids,BRs)是植物中的一類類固醇激素,可調(diào)控許多至關(guān)重要的生長發(fā)育過程。BR信號由細胞質(zhì)膜上的受體BRI1和共受體BAK1的胞外結(jié)構(gòu)域共同感知,BRI1和BAK1都是單次跨膜的富含亮氨酸重復序列的類受體蛋白激酶(leucine-rich repeat receptor-like protein kinase,LRR-RLKs)。當BR信號缺失時,其受體BRI1的激酶活性受負調(diào)因子BKI1的抑制;當BR信號存在時,其可與BRI1和BAK1的胞外結(jié)構(gòu)域結(jié)合,引起B(yǎng)KI1的解離,進而導致BRI1與BAK1的激酶結(jié)構(gòu)域相互結(jié)合和相互磷酸化激活。BRI1-BL-BAK1復合體可起始BR早期信號的感知并激活下游信號的級聯(lián)反應(yīng)。篩選某特異基因的不同突變體,并對這些突變體產(chǎn)生表型的不同分子機制進行深入研究,將有助于發(fā)掘該基因更細致的分子生物學功能。在過去的二十年中,科學家們已篩選到了20多種不同類型的bri1突變體,這些突變體對BRI1不同結(jié)構(gòu)域的功能研究做出了巨大貢獻。其中的一些弱突變體,如bri1-119,bri1-5和bri1-9,還作為非常優(yōu)異的遺傳篩選材料,對BR合成、代謝和信號轉(zhuǎn)導,乃至與其他通路互作調(diào)控元件的發(fā)掘做出了重要貢獻。為了對BRI1調(diào)控BR信號轉(zhuǎn)導的分子機制進行更深入的探究,本研究對TILLING(targeted induced local lesions in genomes)技術(shù)誘導的所有可能的BRI1突變點進行了全面的篩選。我們從ABRC(Arabidopsis Biological Resource Center)訂購了所有87個BRI1 TILLING有義突變的原始種子。通過dCAPs鑒定發(fā)現(xiàn)僅有83個真正含有BRI1突變。由于TILLING誘變的原始背景Col-0中引入了er的突變,加之高劑量的EMS處理可能導致了除BRI1外其它基因的突變,進而導致植物表現(xiàn)出多種非BRI1突變引起的表型。為了排除這些干擾,我們將這些突變體與Col-0至少回交了三代。每一代都用dCAPs的方法鑒定其BRI1的突變。回交三代后,我們發(fā)現(xiàn)大部分突變都沒有造成顯著的表型,只篩選到了9個具有不同程度BR缺陷表型的新型bri1突變體,包括4個微弱突變體(bri1-705,bri1-706,bri1-710和bri1-711),1個弱突變體(bri1-702)和4個強突變體(bri1-703,bri1-704,bri1-708和bri1-709)。同時,我們還對這些突變體產(chǎn)生表型的分子機制進行了深入研究。結(jié)果表明,bri1-702是目前為止BRI1活化環(huán)中發(fā)現(xiàn)的唯一一個弱突變體,bri1-702蛋白在體外的自磷酸化活性降低,且其體內(nèi)的BAK1磷酸化對BL的響應(yīng)敏感性也有所降低。bri1-705是一個胞外的突變體,研究發(fā)現(xiàn)超表達mBAK并不能對其造成強烈的顯性負效應(yīng),說明bri1-705的突變可能影響了BRI1和BAK1在胞外的相互作用。進一步的分子動力學結(jié)構(gòu)模擬研究表明該突變很有可能影響了BRI1、BL和BAK1兩兩之間的氫鍵的形成。本研究中還發(fā)現(xiàn)了一個非常有趣的胞外突變體bri1-706,其表型非常微弱,但其根生長卻像bri1強突變體一樣對BL幾乎不響應(yīng)。這種對BL處理的生理響應(yīng)狀況與植物缺陷表型不一致的bri1突變體還屬首次發(fā)現(xiàn)。以前的報道中,bri1-301在體外檢測不到激酶活性。但在本研究中,我們發(fā)現(xiàn)bri1-301在體內(nèi)仍然有激酶活性,這一發(fā)現(xiàn)澄清了之前人們關(guān)于激酶活性對BRI1的功能可能不重要的錯誤觀點。本文的結(jié)論對BR信號轉(zhuǎn)導早期事件分子細節(jié)的研究具有一定貢獻。本研究中發(fā)現(xiàn)的新型突變體可作為遺傳材料對BR通路的其他調(diào)控元件進行進一步的篩選,這些新型bri1的突變體信息也可以對植物中其它RLKs的研究提供重要參考。
[Abstract]:Brassinosteroids (BRs) is a class of steroid hormones in plants, which can regulate a number of critical growth and development processes.BR signals are shared by the extracellular domain of the receptor BRI1 on the cytoplasmic membrane and the co receptor BAK1. Both BRI1 and BAK1 are the single transmembrane rich leucine repeat receptor protein kinase (Leucin). E-rich repeat receptor-like protein kinase, LRR-RLKs). When the BR signal is missing, the kinase activity of its receptor BRI1 is inhibited by the negative modulation factor BKI1; when the BR signal exists, it can be combined with the extracellular domain of BRI1 and BAK1, resulting in the dissociation, which leads to the combination of the kinase domain and the activation of the phosphorylation of the kinase domain. The -BL-BAK1 complex initiates the perception of early BR signals and activates cascade reactions of downstream signals. Screening the different mutants of a specific gene and studying different molecular mechanisms for the phenotype of these mutants will help to explore the more detailed molecular biological function of the gene. In the past twenty years, scientists More than 20 different types of BRI1 mutants have been screened. These mutants have made great contributions to the functional study of different domains of BRI1. Some of these weak mutants, such as bri1-119, bri1-5 and bri1-9, also serve as excellent genetic screening materials for BR synthesis, metabolism and signal transduction, and even interact with other pathways. In order to further explore the molecular mechanism of the BRI1 regulation of BR signal transduction, this study has made a comprehensive screening of all possible BRI1 mutations induced by the TILLING (targeted induced local lesions in genomes) technology. There were 87 primordial seeds with a sense mutation of BRI1 TILLING. By dCAPs, only 83 were found to contain BRI1 mutations. The mutation of ER was introduced in the original background Col-0 of TILLING mutation, and the high dose of EMS treatment could lead to the mutation of other genes except BRI1, thus leading plants to show a variety of non BRI1 mutations. In order to eliminate these disturbances, we have crossed these mutants with Col-0 at least three generations. Each generation uses dCAPs to identify the mutation of its BRI1. After three generations of backcross, we found that most of the mutations did not cause a significant phenotype, and only 9 new BRI1 mutants with different degrees of BR defect were screened, including 4 microsatellites. Weak mutants (bri1-705, bri1-706, bri1-710 and bri1-711), 1 weak mutants (bri1-702) and 4 strong mutants (bri1-703, bri1-704, bri1-708 and bri1-709). Meanwhile, we have also studied the molecular mechanism of the phenotype of these mutants. The results show that bri1-702 is the only weak process found in the BRI1 activation ring so far. The autophosphoridification activity of bri1-702 protein in vitro decreased and the response sensitivity of BAK1 phosphorylation in the body decreased to BL, and.Bri1-705 was an extracellular mutant. The study found that the overexpression of mBAK could not cause a strong dominant negative effect, indicating that the mutation of bri1-705 may affect the interaction of BRI1 and BAK1 in the extracellular. Further molecular dynamics structural simulation studies show that the mutation is likely to affect the formation of hydrogen bonds between BRI1, BL and BAK1 22. In this study, a very interesting extracellular mutation, bri1-706, was found to be very weak, but its root growth was almost no response to BL as a strong BRI1 mutant. This to BL The BRI1 mutant, which is not consistent with the phenotype of Plant defects, is first discovered. In the previous report, bri1-301 did not detect the kinase activity in vitro. But in this study, we found that bri1-301 still has kinase activity in the body. This discovery clarifies that the function of kinase activity to BRI1 may not be found before. The conclusions of this paper contribute to the study of molecular details of the early event of BR signal transduction. The new mutant found in this study can be used as a genetic material to further screen the other regulatory elements of the BR pathway, and the mutants of these new BRI1 can also be used to study other RLKs in plants. For an important reference.
【學位授予單位】：蘭州大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：Q943.2

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相關(guān)期刊論文 前3條

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本文編號：1986238