本文選題：擬南芥 + 油菜素甾醇��； 參考：《蘭州大學(xué)》2016年博士論文

【摘要】：油菜素甾醇(brassinosteroids,BRs)是一類植物特有的內(nèi)源性甾醇激素,它在植物營養(yǎng)生長和生殖生長的各個方面都發(fā)揮著重要的生理作用。BRs由細(xì)胞膜上受體BRI1和共受體BAK1組成的復(fù)合體感受并起始信號轉(zhuǎn)導(dǎo),經(jīng)細(xì)胞質(zhì)內(nèi)一系列的磷酸化/去磷酸化活動級聯(lián)放大信號至細(xì)胞核內(nèi)轉(zhuǎn)錄因子BES1/BZR1以調(diào)控基因表達(dá)。共受體BAK1及其所屬的SERK家族的其它四個成員均是富亮氨酸重復(fù)類受體激酶(LRR-RLKs)。目前的研究表明BAK1與其它SERKs成員參與BR信號轉(zhuǎn)導(dǎo)、光依賴的細(xì)胞死亡、花藥發(fā)育、植物固有免疫以及氣孔分化等多個生理活動。而對BAK1的兩個突變體elg和bak1-5的研究,進(jìn)一步指出BAK1功能具有復(fù)雜的多樣性,即便是在研究最深入的BR信號轉(zhuǎn)導(dǎo)中它也可能存在未知的功能。此外,鑒于BR信號轉(zhuǎn)導(dǎo)的早期過程即細(xì)胞膜附近的信號傳遞活動仍有諸多細(xì)節(jié)不甚清楚,故我們擬以BAK1為出發(fā)點繼續(xù)深入研究。本研究首先以bak1-4為遺傳背景建立T-DNA插入庫,并篩選可能的與BAK1一起共同介導(dǎo)BR信號或其它信號途徑的功能組分。其間獲得一個典型的BR不敏感突變體128-12-T01,T-DNA側(cè)翼序列克隆后發(fā)現(xiàn)它是TWD1的一個新突變體故重命名為twd1-4。同時另一個點突變體twd1-5從EMS誘變庫中篩選到并被用于后續(xù)研究中。twd1-5對外源施加的油菜素內(nèi)酯(brassinolide,BL)不敏感而對特異的合成抑制劑超敏感,說明它也是BR不敏感型突變體。雜交實驗顯示TWD1不直接參與BRs合成,而是位于BR合成通路的下游。盡管TWD1定位于內(nèi)質(zhì)網(wǎng)并參與ABCB1和ABCB19的轉(zhuǎn)運(yùn),但我們發(fā)現(xiàn)超表達(dá)TWD1不能恢復(fù)bri1的表型。而且twd1-5中BRI1的細(xì)胞膜定位及其蛋白穩(wěn)定性均未改變,即是說TWD1并未涉及BRI1的內(nèi)質(zhì)網(wǎng)質(zhì)量控制活動。而同時作為膜蛋白的TWD1,經(jīng)生化實驗證實它在細(xì)胞膜上與受體BRI1相互作用。twd1-5中BR信號轉(zhuǎn)導(dǎo)受阻,是由于該缺失導(dǎo)致BL誘導(dǎo)的BRI1和BAK1磷酸化顯著減少。此外,TWD1的缺失同時也導(dǎo)致BL誘導(dǎo)的BRI1、BAK1間相互作用減弱故而影響整個BR信號的起始。遺傳及生化證據(jù)指明TWD1通過與BRI1相互作用參與BR信號轉(zhuǎn)導(dǎo)的早期過程,起到維持上游信號傳遞的作用。隨后為進(jìn)一步尋找BR信號通路相關(guān)基因,我們從已有的研究中分析篩選出44個BR相關(guān)基因并將其超表達(dá)到合成突變體br6ox2中觀察其表型改變。其中BED1基因超表達(dá)后使br6ox2葉片變小、植株變矮、果莢縮短,隨后在Col-0中也重現(xiàn)到類似表型。BED1超表達(dá)材料(BED1-OX)的幼苗對BR處理有輕微的不敏感,其成體植株呈現(xiàn)明顯的BR缺失突變體表型。BED1-OX同時也具有典型的erecta突變體表型,包括成簇的花序、果莢變短變寬及果柄縮短等;但未發(fā)現(xiàn)與erecta類似的氣孔發(fā)育缺陷。人工微RNA(amiRNA)技術(shù)構(gòu)建的BED1缺失突變體并無明顯表型而超表達(dá)BED1的同源基因BEDL2、BEDL3呈現(xiàn)與BED1-OX類似的表型,說明它們的功能冗余。深入分析發(fā)現(xiàn),持續(xù)激活MAPK途徑的組分YODA、MKK4和MKK5可以將BED1-OX植株恢復(fù)至野生型表型。這些結(jié)果說明BED1采用和ERECTA類似的MAPK途徑,可能通過抑制細(xì)胞增殖和促進(jìn)分化來調(diào)控植物花序發(fā)育,而這一過程很可能需要受體ERECTA和其共受體BAK1(SERKs)的參與。此外使用全長cDNA超表達(dá)捕獲技術(shù)(FOX hunting system)在bri1-9背景下篩選恢復(fù)型突變體時我們獲得一株莖端分生組織增生的突變體cil9-s1�；蚩寺〖爸噩F(xiàn)實驗確定其為過量表達(dá)RNA聚合酶非催化亞基NRP(B/D/E)6A所致。超表達(dá)NRP(B/D/E)6A及同源基因NRP(B/E)6B不影響RNA聚合酶IV和V的功能,但卻產(chǎn)生多種不穩(wěn)定且類似RNA聚合酶II的相關(guān)缺失突變體nrpb2-1的表型;但這些表型與BR合成及信號轉(zhuǎn)導(dǎo)可能并無直接聯(lián)系。NRP(B/D/E)6A和NRP(B/E)6B各自單突變體沒有明顯表型,但其雙突體則出現(xiàn)胚胎發(fā)育滯后、胚柄異常膨大等多種缺陷以致最終胚胎死亡。這些結(jié)果指出NRP(B/D/E)6A在植物的分生組織和胚胎發(fā)育過程中起著至關(guān)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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q946
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本文編號：1904299