拟南芥中bri1新型TILLING突变体的筛选及其机理研究

发布时间：2018-06-06 10:38

本文选题：油菜素甾醇 + 油菜素内酯　；参考：《兰州大学》2017年博士论文

【摘要】：油菜素甾醇(brassinosteroids,BRs)是植物中的一类类固醇激素,可调控许多至关重要的生长发育过程。BR信号由细胞质膜上的受体BRI1和共受体BAK1的胞外结构域共同感知,BRI1和BAK1都是单次跨膜的富含亮氨酸重复序列的类受体蛋白激酶(leucine-rich repeat receptor-like protein kinase,LRR-RLKs)。当BR信号缺失时,其受体BRI1的激酶活性受负调因子BKI1的抑制;当BR信号存在时,其可与BRI1和BAK1的胞外结构域结合,引起BKI1的解离,进而导致BRI1与BAK1的激酶结构域相互结合和相互磷酸化激活。BRI1-BL-BAK1复合体可起始BR早期信号的感知并激活下游信号的级联反应。筛选某特异基因的不同突变体,并对这些突变体产生表型的不同分子机制进行深入研究,将有助于发掘该基因更细致的分子生物学功能。在过去的二十年中,科学家们已筛选到了20多种不同类型的bri1突变体,这些突变体对BRI1不同结构域的功能研究做出了巨大贡献。其中的一些弱突变体,如bri1-119,bri1-5和bri1-9,还作为非常优异的遗传筛选材料,对BR合成、代谢和信号转导,乃至与其他通路互作调控元件的发掘做出了重要贡献。为了对BRI1调控BR信号转导的分子机制进行更深入的探究,本研究对TILLING(targeted induced local lesions in genomes)技术诱导的所有可能的BRI1突变点进行了全面的筛选。我们从ABRC(Arabidopsis Biological Resource Center)订购了所有87个BRI1 TILLING有义突变的原始种子。通过dCAPs鉴定发现仅有83个真正含有BRI1突变。由于TILLING诱变的原始背景Col-0中引入了er的突变,加之高剂量的EMS处理可能导致了除BRI1外其它基因的突变,进而导致植物表现出多种非BRI1突变引起的表型。为了排除这些干扰,我们将这些突变体与Col-0至少回交了三代。每一代都用dCAPs的方法鉴定其BRI1的突变。回交三代后,我们发现大部分突变都没有造成显著的表型,只筛选到了9个具有不同程度BR缺陷表型的新型bri1突变体,包括4个微弱突变体(bri1-705,bri1-706,bri1-710和bri1-711),1个弱突变体(bri1-702)和4个强突变体(bri1-703,bri1-704,bri1-708和bri1-709)。同时,我们还对这些突变体产生表型的分子机制进行了深入研究。结果表明,bri1-702是目前为止BRI1活化环中发现的唯一一个弱突变体,bri1-702蛋白在体外的自磷酸化活性降低,且其体内的BAK1磷酸化对BL的响应敏感性也有所降低。bri1-705是一个胞外的突变体,研究发现超表达mBAK并不能对其造成强烈的显性负效应,说明bri1-705的突变可能影响了BRI1和BAK1在胞外的相互作用。进一步的分子动力学结构模拟研究表明该突变很有可能影响了BRI1、BL和BAK1两两之间的氢键的形成。本研究中还发现了一个非常有趣的胞外突变体bri1-706,其表型非常微弱,但其根生长却像bri1强突变体一样对BL几乎不响应。这种对BL处理的生理响应状况与植物缺陷表型不一致的bri1突变体还属首次发现。以前的报道中,bri1-301在体外检测不到激酶活性。但在本研究中,我们发现bri1-301在体内仍然有激酶活性,这一发现澄清了之前人们关于激酶活性对BRI1的功能可能不重要的错误观点。本文的结论对BR信号转导早期事件分子细节的研究具有一定贡献。本研究中发现的新型突变体可作为遗传材料对BR通路的其他调控元件进行进一步的筛选,这些新型bri1的突变体信息也可以对植物中其它RLKs的研究提供重要参考。
[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.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2

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