【作者】 许瑞瑞；

【导师】 郑成超；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 植物在生长和发育的整个生命周期中,面临着许多不利的外部环境,比如土壤盐害、干旱、涝害、温度胁迫、各种离子胁迫等。在长期的进化过程中,植物形成了响应胁迫和提高抗性的多种适应机制,这些适应机制通常都有成百上千种基因表达所控制。RNA解旋酶是在RNA复制过程中通过解开氢键进而催化双链RNA解旋的酶,其主要功能是利用水解ATP产生的能量修饰RNA的结构。RNA很容易形成稳定却没有功能的二级结构,因此它们需要通过RNA伴侣来行使正确的功能。RNA解旋酶能够利用ATP水解产生的能量,有效的阻止RNA的错误折叠,使RNA正确折叠后行使正确的功能,所以RNA解旋酶被认为是RNA的最佳伴侣。在不同的生物体中以及不同的器官中,都己经证实RNA解旋酶的存在,而且总是起着十分重要的作用。RNA解旋酶可以调控并影响RNA转录、mRNA剪切、蛋白质翻译、RNA降解、核糖体发生和组装、配子及胚胎发生、以及细胞生长和分化等生命活动。近年来,动物和植物中的RNA解旋酶已成为本领域的研究热点。研究表明,RNA解旋酶在植物多种生长发育以及生理过程中起到关键调控作用。2001年,Seki等首次在拟南芥中发现冷害诱导型DEAD-box解旋酶基因AB050574;DEAD-box RNA解旋酶CRYOPHYTE/LOS4定位于核膜上,可以调节拟南芥的耐寒能力,而且对于mRNA的输出和植株的生长发育以及逆境响应也具有重要的作用;STRS1和STRS2是两个胁迫响应抑制子,可以负调控拟南芥对多种非生物胁迫的响应。在胁迫应答途径中,RNA解旋酶位于逆境胁迫应答途径的上游,在转录水平、转录后水平或翻译水平上调控下游功能基因的表达。拟南芥RNA解旋酶家族是一个很大的家族,成员繁多,功能复杂,研究较多的属于DEAD-box家族成员,但对于拟南芥DExD/H-box RNA解旋酶与逆境胁迫及生长发育之间的关系,仍不是很清楚。在模式植物拟南芥中,我们分离鉴定了DExD/H-box家族的两个成员AtHELPS和AtRDEM,并分别对其表达模式和功能进行了分析:(1)拟南芥RNA解旋酶AtHELPS蛋白靠近氨基酸N段区域中包含RNA解旋酶所特有的八个保守基序,I,Ia,Ib,II,III,IV,V和VI,具有解旋酶家族蛋白的基本特征。(2)利用洋葱表皮细胞进行的瞬时表达实验证明AtHELPS非特异性地定位于整个细胞中,并且在细胞质膜和细胞核中的表达量稍高于细胞质中。对转AtHELPS-GFP融合蛋白的稳定表达植株的研究结果证实了AtHELPS的表达模式,该结果与瞬时表达实验结果一致。(3)利用实时定量RT-PCR和GUS染色实验证明,AtHELPS主要在幼苗时期的根和叶脉的维管束组织中表达,在成苗时期的根中也有较强的表达;利用切片实验观察到该基因主要在中柱鞘细胞和靠近木质部的薄壁细胞中表达。AtHELPS的表达受到多种非生物胁迫的调控,包括盐胁迫、低钾、冷害等,其中受低钾胁迫最明显。另外除了玉米素能诱导其表达外,其他激素对该基因的表达量并无明显影响。(4)通过对三种基因型(WT,helps和OE6)的芯片杂交实验所得数据的GO分析,证明在正常条件下,三个基因型之间的差异表达基因中有近半数编码转录因子,它们分别属于bHLH、WRKY、MYB转录因子等家族以及编码一些蛋白激酶类,说明该基因在调节环境胁迫诱导基因表达方面有重要的作用,很可能参与了某种或多种生物胁迫过程。另外在表达差异基因中,还有一部分属于离子结合蛋白和离子通道类蛋白,这也说明该基因在离子运输和转运中也发挥功能。(5)AtHELPS调节拟南芥植株对盐胁迫的耐受性。AtHELPS突变体和超表达植株表现出了对盐胁迫敏感性的不同。正常生长条件下,二者与WT相似,没有明显的表型差异。在盐胁迫下,OE6超表达植株表现出对NaCl高敏感性,而helps突变体则表现出一定的盐胁迫耐受性。通过对耐盐基因和转录因子的qRT-PCR分析,盐胁迫条件下,在helps突变体,超表达株系和WT植株中,SOS1,SOS2等基因的表达并没有明显差异,而WRKY转录因子家族成员WRKY25和WRKY33的表达量在三种基因型中的变化差异较大,说明AtHELPS并不参与影响SOS调控途径,它可能在调节WRKY转录因子家族WRKY25和WRKY33的表达时有着重要的作用,进而影响拟南芥对盐胁迫的耐受性。非损伤微测结果显示,不管是正常条件还是盐胁迫条件下,各个基因型植株中的Na+流速并没有明显的变化,暗示AtHELPS可能是影响了盐胁迫条件下其它的离子运输过程。(6)AtHELPS调节拟南芥植株对低钾胁迫的耐受性。AtHELPS突变体和超表达植株表现出了对低钾胁迫耐受性的不同。在正常的生长条件下,突变体和超表达植株幼苗和成苗的生长和形态上的发育都与野生型植株没有明显差异。低钾条件下,WT和超表达株系OE6的种子萌发及生长要明显滞后于突变体helps。qRT-PCR实验结果显示,拟南芥受到低钾胁迫时AtHELPS影响了AKT1,CBL1,CBL9和CIPK23的表达,AKT1表达受到诱导后,介导胞外钾离子向细胞内流动。通过非损伤微测技术获得的数据分析表明,在正常条件下,三者之间的K~+外流没有明显的差异;然而在钾缺乏的条件下,突变体helps幼苗根的分生组织中受诱导的K~+内流比WT和OE6的大,表明AtHELPS可能通过调控钾离子通道AKT1的表达参与了拟南芥钾缺乏时K~+的离子运输过程。(7)AtRDEM在拟南芥生长发育过程中起作用。超表达AtRDEM植株表现出株型变小、莲座叶数目少、顶端优势不明显甚至缺失、茎短、花发育畸形、果荚成簇、植株矮小、茎分枝多等等发育不正常的表型,说明AtRDEM调控拟南芥的生长发育。通过对基因芯片结果的分析,AtRDEM的超表达使很多生物学过程发生了变化。其中,植物发育相关基因和激素响应途径明显受到影响。因此,拟南芥AtRDEM可能通过参与对调控生长发育有重要作用的某种激素代谢过程,影响了拟南芥的生长发育。通过对芯片结果中发育相关和激素响应相关的关键基因的qRT-PCR验证分析,结果表明与芯片数据是基本一致的。另外,我们还发现合成独脚金萌发素内酯的关键酶基因CCD7和CCD8在OEAtRDEM株系中表达量明显上调,而处于合成下游的Cyt P450的表达量在各超表达株系是明显下调的。这说明在超表达拟南芥植株中独脚金萌发素内酯合成的量有可能减少,推测可能是超表达AtRDEM基因后影响了合成酶基因的表达,调节了该激素的合成和信号转导途径,最终与其他因素共同导致超表达株系出现分枝增多,顶端优势不明显甚至缺失等发育异常的表型。更多还原

【Abstract】 Plants are sessile organisms and hence cannot escape unfavorable environmental conditions within their life cycle, such as high salinity, drought, waterlog, temperature stress, various ion stresses and so on. To deal with the abiotic stresses, plants have developed a series of coordinated responses involving a complex variety of tolerance mechanisms that are activated by the expression of relevant genes.RNA helicases refer to enzymes that use energy derived from the hydrolysis of a nucleotide triphosphate to unwind double-stranded RNAs and modify RNA structure. As RNA molecules are prone to forming stable nonfunctional secondary structures, their proper function requires RNA chaperones. RNA helicases are prominent candidates for RNA chaperones because they can actively disrupt misfolded RNA structures to ensure correct folding. Helicases belong to a class of molecular motor proteins in yeast, animals and plants. RNA helicases are proved to be involved in every step of RNA metabolism, including nuclear transcription, pre-mRNA splicing, translation, RNA decay, ribosome biogenesis and assembly, nucleocytoplasmic transport, embryogenesis, cell division and differentiation. Because of their multiple functions, RNA helicases have been the research hotpot in life sciences.Several studies have shown the majority of RNA helicases is involved in plant growth and development and diverse physiological processes. In 2001, Seki et al. reported firstly that an Arabidopsis helicase gene AB050574 was induced by cold treatment, suggesting that helicases might play a role in plant stress response. Recently, an Arabidopsis DEAD box RNA helicase LOS4 localized in the cytoplasm and enriched at the nuclear rim was shown to be essential for mRNA export and important for development and stress responses in Arabidopsis. Another two DEAD box RNA helicases, STRS1 and STRS2, were shown to improve Arabidopsis responses to multiple abiotic stresses as negative regulators, such as salt, osmotic stress, heat stress and ABA. In response to abiotic stress, RNA helicase have been proved to regulate downstream genes expression on transcription, post-transcription and translation level. In Arabidopsis, RNA helicase is the biggest family, with many members and various functions. Nevertheless, the relationships between DExD/H-box RNA helicase and environmental stress and development are still largely unknown in Arabidopsis.Two DExD/H-box RNA helicases have been isolated and characterized from the Arabidopsis genome, which were designated as AtHELPS (AT3G46960) and AtRDEM (AT5G47010). Their expression pattern and function are analyzed in detail.(1) Arabidopsis RNA helicase AtHELPS possesses eight conserved motifs, I, Ia, Ib, II, III, IV, V and VI in N terminal; these characterizations indicate that AtHELPS protein has hallmarks of DExD/H-box RNA helicase.(2) Transient analysis with onion epidermal cells and stable analysis with AtHELPS-GFP transgenic lines by Confocal Microscopy revealed that AtHELPS is unexclusively localized in the whole cell, but higher expression level is detected in plasma membrane and nucleus than in cytoplasm.(3) By quantitative real-time reverse transcription-PCR (qRT-PCR) and promoter:β-glucouronidase (GUS) fusions analysis, AtHELPS is shown to mainly express in vascular tissues of leaves and roots in young seedlings. The expression of AtHELPS could be regulated by several stresses like salt, low K~+ and low temperature stress, especially by low K~+ stress. Moreover, the expression level of AtHELPS could be induced by zeatin but not by other hormones.(4) GO analysis based on the microarray data indicated that around half amount of genes with different expression patterns encode the protein kinase and transcription factors such as bHLH, WRKY, MYB etc. Among those differentially expressed genes, a certain amount of which encodes ion binding proteins, ion channel proteins and transporters. These data suggest that AtHELPS might participate in the abiotic stress responses and ion transportation.(5) AtHELPS regulated Arabidopsis responses and tolerances to salt stress. Our results indicate that helps mutant, OE6 and WT plants have different sensitivity to salt stress. In our experiments, we discovered both seedling and adult of helps mutant and OE6 plants showed no morphological or developmental differences compared to WT when grown under normal conditions. But the results suggest that the helps mutant plants are more tolerant to salt stress, whereas the OE6 plants are more sensitive to salt stress. qRT-PCR analysis showed that the expression levels of WRKY25 and WRKY33 were higher in the OE6 than those in wild-type and helps mutant plants under salt stress condition, but the expression of SOS family genes were not affected. We thus suggest that the DEVH box RNA helicase AtHELPS might be involved in the regulation of expression of WRKY25 and WRKY33 under salt stress condition. We also applied noninvasive ion-selective microelectrode ion flux measurements to clarify genotype differences of Na+ flux profiles from root meristem zones of Arabidopsis. The net Na+ flux in helps mutant, wild type and OE6 seedlings showed no difference under salt stress condition compared to normal condition, suggesting that AtHELPS might not be involved in regulating Na+ uptake and accumulation in Arabidopsis roots, but possibly involved in other ion uptake, assimilation and transportation.(6) AtHELPS regulated Arabidopsis responses and tolerances to low K~+ stress. Our results indicate that helps mutant, OE6 and WT plants have different sensitivity to low K~+ stress. The seed germination percentage and seedling fresh weight of the helps mutants were higher than those of wide-type and OE6 plants in the low K~+ condition, whereas no differences were observed among the three genotypes under normal conditions. Interestingly, qRT-PCR analysis showed that the expression of AKT1, CBL1/9, and CIPK23 in the helps mutants were consistently higher than those in WT and OE6 plants after low K~+ treatment. We thus suggest that the DEVH box RNA helicase AtHELPS might be involved in the regulation of the AKT1-mediated and CBL/CIPK-regulated K~+ uptake pathway under the low K~+ stress condition. We also applied noninvasive ion-selective microelectrode ion flux measurements to clarify genotype differences of K~+ flux profiles from root meristem zones of Arabidopsis. The net K~+-induced influx in helps mutants was higher than that of wild type and OE6 seedlings when they were exposed to potassium deprivation, suggesting that AtHELPS might be involved in regulating K~+ uptake in Arabidopsis roots via high-affinity transporters like AKT1.(7) AtRDEM plays an important role in the growth and development of Arabidopsis. OEAtRDEM exhibited obvious phenotypic alterations, such as lacked an apical dominance, shorter stem, flower abnormality, silique cluster and more shoot branching. These data reveal that AtRDEM plays an important role in plant development in Arabidopsis. By microarray analysis, many biological processes are affected by overexpressing AtRDEM, such as developmental progress, hormone metabolic pathway, response to abiotic or biotic stimulus, signal transduction etc. Among these changed genes, the expressions of genes involved in development, hormone responsive and metabolic are changed significantly in AtRDEM overexpression plants compared to WT plants, suggesting AtRDEM mediated Arabidopsis growth and development, most probably through hormone. In addition, we found that the expression level of Carotenoid Cleavage Dioxygenase 7 (CCD7) and Carotenoid Cleavage Dioxygenase 8 (CCD8) were enhanced in AtRDEM overexpression lines, but the cytochrome P450 monooxygenase located downstream of the strigolactone biosynthesis pathway was reduced. These results suggested that the novel hormone strigolactone might not accumulate and move acropetally to inhibit axillary bud outgrowth. Together, we speculate that the AtHELPS may be involved in regulating the strigolactone biosynthesis and signal transduction pathway.更多还原