拟南芥TWD1调节油菜素甾醇信号转导通路的早期过程

发布时间：2018-05-18 04:08

  本文选题：拟南芥 + 油菜素甾醇　； 参考：《兰州大学》2016年博士论文

【摘要】：油菜素甾醇(brassinosteroids,BRs)是一类植物特有的内源性甾醇激素,它在植物营养生长和生殖生长的各个方面都发挥着重要的生理作用。BRs由细胞膜上受体BRI1和共受体BAK1组成的复合体感受并起始信号转导,经细胞质内一系列的磷酸化/去磷酸化活动级联放大信号至细胞核内转录因子BES1/BZR1以调控基因表达。共受体BAK1及其所属的SERK家族的其它四个成员均是富亮氨酸重复类受体激酶(LRR-RLKs)。目前的研究表明BAK1与其它SERKs成员参与BR信号转导、光依赖的细胞死亡、花药发育、植物固有免疫以及气孔分化等多个生理活动。而对BAK1的两个突变体elg和bak1-5的研究,进一步指出BAK1功能具有复杂的多样性,即便是在研究最深入的BR信号转导中它也可能存在未知的功能。此外,鉴于BR信号转导的早期过程即细胞膜附近的信号传递活动仍有诸多细节不甚清楚,故我们拟以BAK1为出发点继续深入研究。本研究首先以bak1-4为遗传背景建立T-DNA插入库,并筛选可能的与BAK1一起共同介导BR信号或其它信号途径的功能组分。其间获得一个典型的BR不敏感突变体128-12-T01,T-DNA侧翼序列克隆后发现它是TWD1的一个新突变体故重命名为twd1-4。同时另一个点突变体twd1-5从EMS诱变库中筛选到并被用于后续研究中。twd1-5对外源施加的油菜素内酯(brassinolide,BL)不敏感而对特异的合成抑制剂超敏感,说明它也是BR不敏感型突变体。杂交实验显示TWD1不直接参与BRs合成,而是位于BR合成通路的下游。尽管TWD1定位于内质网并参与ABCB1和ABCB19的转运,但我们发现超表达TWD1不能恢复bri1的表型。而且twd1-5中BRI1的细胞膜定位及其蛋白稳定性均未改变,即是说TWD1并未涉及BRI1的内质网质量控制活动。而同时作为膜蛋白的TWD1,经生化实验证实它在细胞膜上与受体BRI1相互作用。twd1-5中BR信号转导受阻,是由于该缺失导致BL诱导的BRI1和BAK1磷酸化显著减少。此外,TWD1的缺失同时也导致BL诱导的BRI1、BAK1间相互作用减弱故而影响整个BR信号的起始。遗传及生化证据指明TWD1通过与BRI1相互作用参与BR信号转导的早期过程,起到维持上游信号传递的作用。随后为进一步寻找BR信号通路相关基因,我们从已有的研究中分析筛选出44个BR相关基因并将其超表达到合成突变体br6ox2中观察其表型改变。其中BED1基因超表达后使br6ox2叶片变小、植株变矮、果荚缩短,随后在Col-0中也重现到类似表型。BED1超表达材料(BED1-OX)的幼苗对BR处理有轻微的不敏感,其成体植株呈现明显的BR缺失突变体表型。BED1-OX同时也具有典型的erecta突变体表型,包括成簇的花序、果荚变短变宽及果柄缩短等;但未发现与erecta类似的气孔发育缺陷。人工微RNA(amiRNA)技术构建的BED1缺失突变体并无明显表型而超表达BED1的同源基因BEDL2、BEDL3呈现与BED1-OX类似的表型,说明它们的功能冗余。深入分析发现,持续激活MAPK途径的组分YODA、MKK4和MKK5可以将BED1-OX植株恢复至野生型表型。这些结果说明BED1采用和ERECTA类似的MAPK途径,可能通过抑制细胞增殖和促进分化来调控植物花序发育,而这一过程很可能需要受体ERECTA和其共受体BAK1(SERKs)的参与。此外使用全长cDNA超表达捕获技术(FOX hunting system)在bri1-9背景下筛选恢复型突变体时我们获得一株茎端分生组织增生的突变体cil9-s1。基因克隆及重现实验确定其为过量表达RNA聚合酶非催化亚基NRP(B/D/E)6A所致。超表达NRP(B/D/E)6A及同源基因NRP(B/E)6B不影响RNA聚合酶IV和V的功能,但却产生多种不稳定且类似RNA聚合酶II的相关缺失突变体nrpb2-1的表型;但这些表型与BR合成及信号转导可能并无直接联系。NRP(B/D/E)6A和NRP(B/E)6B各自单突变体没有明显表型,但其双突体则出现胚胎发育滞后、胚柄异常膨大等多种缺陷以致最终胚胎死亡。这些结果指出NRP(B/D/E)6A在植物的分生组织和胚胎发育过程中起着至关重要的作用。
[Abstract]:Brassinosteroids (BRs) is a kind of plant specific endogenous steroid hormone. It plays an important physiological role in all aspects of plant nutrition growth and reproductive growth,.BRs is sensed by the complex of receptor BRI1 on cell membrane and co receptor BAK1, and begins to signal transduction. A series of phosphorylation through the cytoplasm. The dephosphorylation cascade amplifies the signal to the nuclear transcription factor BES1/BZR1 to regulate the gene expression. The co receptor BAK1 and the other four members of the SERK family belong to the leucine repeat receptor kinase (LRR-RLKs). The present study shows that BAK1 and other SERKs members are involved in BR signal transduction, light dependent cell death, and flowers. Drug development, plant inherent immunity and stomatal differentiation, and the study of two mutants of BAK1, ELG and bak1-5, further point out that BAK1 function has complex diversity, even in the most in-depth study of BR signal transduction, it may also have unknown functions. In addition, the early process of BR signal transduction is fine. There are still a lot of details about the signal transmission near the membrane, so we should continue to study the BAK1 as a starting point. First, we set up a T-DNA insertion library with the genetic background of bak1-4, and screened the possible functional components of the BR signal or other signal pathways together with BAK1. In the meantime, a typical BR non sensitive group was obtained. The mutant 128-12-T01, T-DNA flanking sequence was cloned and found to be a new mutant of TWD1, so it was renamed twd1-4. and another point mutation, twd1-5, was screened from EMS mutagenesis and was used in the follow-up study..twd1-5 is insensitive to exogenous Brassinolide (brassinolide, BL), and hypersensitivity to specific synthetic inhibitors. It shows that it is also an insensitive mutant of BR. Hybridization experiments show that TWD1 is not directly involved in BRs synthesis, but is located in the downstream of BR synthesis pathway. Although TWD1 is located in endoplasmic reticulum and participates in the transport of ABCB1 and ABCB19, we found that the overexpression TWD1 does not restore the phenotype of BRI1. Moreover, BRI1 cell membrane localization and protein stability in twd1-5 No changes were made, that is, TWD1 did not involve BRI1's endoplasmic reticulum quality control activities. While TWD1, a membrane protein, was confirmed by biochemical experiments that the BR signal transduction in the cell membrane interacting with receptor BRI1 was hindered by the loss of BRI1 and BAK1 phosphorylation induced by BL, and the absence of TWD1 was also due to the loss of TWD1. The interaction between BRI1 and BAK1 induced by BL has weakened the origin of the whole BR signal. Genetic and biochemical evidence indicates that TWD1 participates in the early process of BR signal transduction by interacting with BRI1 and plays the role of maintaining the upstream signal transmission. Subsequently, we have analyzed the genes of the pathway related to the BR signal through the analysis of the existing research. 44 BR related genes were screened and overexpressed in the synthetic mutant br6ox2 to observe the phenotypic changes. The BED1 gene overexpressed the br6ox2 leaves, the plants became shorter, and the pod shortened, and the seedlings of the similar phenotypic.BED1 overexpression material (BED1-OX) were subsequently reproduced in Col-0 with slight insensitivity to BR treatment, and their adult plants The obvious phenotype of BR deletion mutant.BED1-OX also has typical erecta mutant phenotypes, including clusters of inflorescence, fruit pods widening and shortening, but no stomatal development similar to erecta is found. The BED1 deletion mutants constructed by artificial micro RNA (amiRNA) technology have no obvious phenotypes to overexpress the homology of BED1. The gene BEDL2, BEDL3 presents a phenotype similar to that of BED1-OX, indicating their functional redundancy. In-depth analysis shows that the components of the MAPK pathway, YODA, MKK4 and MKK5, can restore the BED1-OX plants to the wild phenotype. These results suggest that BED1 adopts a MAPK pathway similar to ERECTA, which may be mediated by inhibiting cell proliferation and promoting differentiation. Plant inflorescence is controlled, and this process is likely to require the participation of receptor ERECTA and its co receptor BAK1 (SERKs). In addition, a full-length cDNA overexpression capture technique (FOX hunting system) is used to screen a restorer mutant in the bri1-9 background and we obtain a clone and reproduction experiment of a mutant of stem meristem. It is determined that the overexpression of RNA polymerase's non catalytic subunit NRP (B/D/E) 6A. The overexpression of NRP (B/D/E) 6A and homologous gene NRP (B/E) 6B does not affect the function of RNA polymerase IV and reproduction, but produces a variety of unstable and similar deletion mutants similar to that of the polymerase. There is no obvious phenotype in the single mutants of.NRP (B/D/E) 6A and NRP (B/E) 6B, but the BIS process has many defects such as embryonic development lag, abnormal expansion of the embryo and the final embryo death. These results indicate that NRP (B/D/E) 6A plays a vital role in the process of plant meristem and embryo development.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q946
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本文编号：1904299