【作者】 孙清华；

【导师】 张举仁；

【作者基本信息】 山东大学 ， 细胞生物学， 2011， 博士

【摘要】 对高等植物非生物胁迫应答的分子机制的研究具有重要的意义。拟南芥是一种典型的甜土植物,它在耐盐耐早机制研究方面有其自身的局限性。盐芥是拟南芥的近缘种,是一种高度耐盐耐旱的盐生植物,且具有基因组小,cDNA序列与拟南芥相似程度高等优点,已成为人们研究植物非生物胁迫响应的模式植物。基因表达调控发生在染色体、转录、转录后、翻译、翻译后多个水平上,其中转录水平的调控尤为重要。启动子作为控制基因转录的顺式作用因子调控基因转录的起始,在基因表达调控中发挥重要作用,而且其调控作用的实施需要转录因子蛋白的参与。本研究以盐芥液泡焦磷酸酶基因TsVP的启动子为实验材料,通过对该启动子核心元件的鉴定及其上游结合蛋白的功能分析,揭示了TsVP基因对盐胁迫诱导响应的部分机制。本实验室高峰等发现,尽管在酵母和烟草中过表达TsVP和AVP1都可以提高宿主的耐盐性,但它们在盐胁迫条件下表达模式不同：TsVP的表达受盐胁迫诱导,而AVP1则对盐诱导不发生反应。为了研究造成这种差异的原因,本工作克隆出这两个基因的启动子区域并进行生物信息学分析,发现二者在顺式作用元件的种类,数量以及分布存在明显的差异。为了较深入了解TsVP启动子的结构和特性,构建了一系列的5’端缺失突变体,将它们分别与报告基因gus连接,用于转化拟南芥。在转基因拟南芥中,全长TSVP启动子(2200bp)在正常条件下高强度启动报告基囚的表达,其GUS表达强度与CaMV 35S启动子驱动的GUS表达强度差异不大；在盐胁迫下,其GUS表达活性在根部和叶中受到明显的诱导作用,升高到未胁迫时的3倍左右。PT1(全长TsVP启动子连接GUS报告基因)和PA1(全长AVP1启动子连接GUS报告基因)在正常生长条件下具有相似的表达模式,几乎在种子外的每一个组织中都具有活性。但在盐胁迫处理后,PT1在根、叶中的表达受到明显的诱导,在根部高强度表达主要出现在根尖处,而在PA1中未发现这种诱导现象。PT1启动子的高驱动能力表明该启动子在植物基因工程中有很好的应用价值。在5’系列缺失突变体中,PT2与PT1相比缺少了一段856bp的区域(-2200至-1344),导致PT2的活性要明显低于PT1的,我们推测在-2200到-1344这段区域存在着可以明显提高启动子活性的增强子元件。此外,PT2在花中表现出明显的花药特异性表达模式。这种表达模式同样出现在缺失突变体PT3到PT6的转基因植株中,但在PT7到PT9中消失。我们推测一个AAATGA元件可能在花的花药特异表达模式中起关键作用。通过对系列缺失突变体在正常条件以及盐胁迫条件下的GUS基因表达分析,最终鉴定得到一段130bp(-667至-538)的核心序列。该序列对于TsVP启动子的盐胁迫诱导响应具有重要的作用。农杆菌介导的烟草叶片GUS瞬时表达分析也表明这130bp区域可以很好的响应盐胁迫环境。在该区域尚未发现与盐胁迫响应相关的已知作用元件,因此对该区域进行进一步分析,找到其中存在的核心序列,并找到调控该元件的功能蛋白,无疑具有重要的学术意义。以这段核心序列为诱饵,通过酵母单杂交系统筛选得到了与之相互作用的两个蛋白,命名为TsNacl和TsVOZ1。TsNac1的ORF全长918bp,与拟南芥RD26(AT4G27410)具有86%的核苷酸相似性,92%的氨基酸相似性。RT-PCR检测表明,TsNac1基因在正常生长条件下的表达量在根中远小于叶中的,但在盐胁迫、干旱胁迫和ABA处理后,根中表达量的变化幅度高于叶中的。TsNac1基因的表达受盐胁迫、干旱胁迫和ABA胁迫的诱导。在这三种胁迫条件下该基因表达强度的变化幅度有不同,对ABA诱导的响应最明显,尤其是在根中,处理12小时后其表达量上升了1000多倍。在盐胁迫条件下TsNac1基因的表达水平可上调60多倍,而对干旱诱导的响应相对较弱,尤其在叶中。这些结果表明该基因有可能是盐芥耐盐机制的重要成员,与ABA信号传导有重要联系。构建TsNac1原核表达载体并转化大肠杆菌BL21,诱导转化菌表达重组蛋白。利用已获得的TsNac1重组蛋白,与上述130bp的启动子序列进行体外结合试验。EMSA试验的结果表明,TsNac1蛋白可以很好的与该片段在体外结合,而且这种结合具有良好的特异性。鉴于TsNac1与TsVP启动子中的盐诱导响应区域特异性结合,而且其本身的表达受到盐、干旱以及ABA的诱导,推测TsNac1很可能就是我们要寻找的调控TsVP表达的上游转录因子,对其进行深入研究以揭示TsNac1的生物学功能以及其调控的下游基因网络具有重要的意义。通过在拟南芥中过表达和抑制表达TsNac1,初步分析了该基因在植物耐盐性方面的作用。TsNac1基因的过表达提高了转基因拟南芥的耐盐性,而抑制该基因则明显增加了转基因拟南芥的盐敏感性。鉴定TsNac1在TsVP启动子上的结合位点可以为了解其作用机制提供有价值的参考资料,也可以为植物基因工程改良提供具有使用价值的启动子元件,因此鉴定TsNac1在TsVP启动子上的靶位点具有重要的意义。利用不同的过量非标记竞争性DNA来竞争标记DNA与TsNac1蛋白的结合,鉴定出一段20bp的DNA序列(GAATATACCATGGA TAAGC A)为该蛋白的结合位点。在这段DNA序列中包含CATG元件。目前认为NAC家族蛋白的结合位点为CACG,但是Trans等研究表明拟南芥中NAC家族的蛋白也可结合一段MYC-like CATGTG位点。推测CATG可能是TsNac1蛋白结合的核心位点,其结合也需要周围的几个甚至十几个核苷酸序列。利用CHIP-on-chip技术对该蛋白在拟南芥染色体中的结合位点进行了详细的分析,并利用RT-PCR对实验结果进行了验证。结果表明在拟南芥中该蛋白与284个基因的启动子区域有结合作用,这284个基因参与了众多的生物学过程,包括了代谢,发育,细胞定位,刺激响应,生殖,蛋白磷酸化,氧化还原等生物学过程。值得注意的是在这284个基因中,有大约70个属于转录因子类基因,占25%左右,而拟南芥中转录因子只占全部基因数量的5%左右。也就是说在TsNac1的靶基因中,转录因子所占的比例大概是正常比例的5倍,而且其靶基因中包括了像DREB这种公认的与植物抗逆有关的转录因子,这也表明在TsNac1基因在植物抗逆中的重要调控作用。此外该基因还可以通过调节一些小分子转运体基因,结构基因以及一些结合蛋白基因的表达来调控下游功能基因的表达,而且该基因也可以直接调控一些功能基因的表达水平。这些结果表明TsNac1在基因调控网络中应该处于一个较为上游的位置。此外,TsVP在拟南芥中的同源基因AVP1并不在这些靶基因中,通过前面启动子分析的工作我们知道AVP1虽然属于抗逆相关基因,但是其本身的表达并不受盐胁迫的诱导,这一结果表明TsNac1并没有参与AVP1基因的调控,这与我们的预期是相符合的。另外,利用酵母单杂交系统还鉴定出与基于TsVP启动子构建的诱饵相结合的TsVOZ1蛋白。该蛋白与拟南芥中的AVOZ1具有85%的核苷酸相似性,90%的氨基酸相似性,其表达受盐胁迫的诱导,但是却不受干旱以及ABA的诱导,而且其受盐胁迫响应的程度远不如TsNac1明显。根据Mitsuda N等人对AVOZ1的研究结果,我们在TsVP启动子区域中,我们找到了一段GCGTNx7ACGC回文序列,即GCGTCGGCTGCACGC (-274到-259),但是这段序列并不在上述我们鉴定得到的130bp核心序列中。通过在拟南芥中对TsVOZ1基因进行过表达和抑制表达,得出TsVOZ1的过表达以及基因敲除并没有明显影响转基因拟南芥的耐盐性。TsVOZ1虽然与该启动子有结合,然而并没有参与该基因盐胁迫响应的调控。这些工作通过对盐芥中TsVP启动子及其上游调控蛋白的功能分析,不仅为植物基因工程提供具有自主知识产权的调控元件和耐盐基因,而且为植物盐胁机制的深入探讨提供了新资料,可望为我国大面积盐碱地的开发利用做贡献。更多还原

【Abstract】 It is meaningful to obtain more in-depth understanding of the molecular mechanisms of abiotic stress responses in higher plants. Arabidopsis is a salt-sensitive plant. It has some limitations in the salt and drought tolerance mechanism research. Thellungiella halophila, the relative species of Arabidopsis thaliana, is a highly salt-tolerant and drought-tolerant plant. It has the advantages such as having a small genome and sharing high cDNA sequence similarity with Arabidopsis. It is now becoming the model plant for the study of the plant stress response mechanisms.Regulation of gene expression could occur in the chromosome, transcription, post-transcription, translational and post-translational levels. Especially, the regulation in the transcription level is more important. Promoters containing the cis-acting elements play a very important role in the regulation of the gene transcription and the role of this regulation requires the participation of the upstream transcription factor proteins. In this study, the TsVP gene promoter from Thellungiella halophila was studied in detail. The identification of the core promoter region and the functional analysis of its upstream binding protein revealed parts of the salt stress response mechanisms of the TsVP gene.Gao et al found the TsVP and AVP1 genes had different expression profiles under salt stress condition although they had the same salt stress tolerance function in both the yeast and tobacco. The TsVP was salt stress inducible while the AVP1 was not. In order to investigate the reasons, we cloned and analyzed the promoter regions of these two genes, the result indicated that the categories, number and distribution of the cis-acting elements in these two promoters were different.Seried of 5’-deleted promoter-GUS mutants were constructed and transformed into Arabidopsis to study this promoter in detail. For the full-length TsVP promoter (2200bp), it could drive the expression of GUS reporter gene and its activity was almost the same as the well used CaMV 35S promoter under normal condition. While treated with salt stress, its activity was obviously induced up to about 3-fold level both in the leaves and roots. PT1 and PA1 had similar expression pattern under normal condition, but the activity of PT1 was induced especially in the root tips while PA1 was not. PT1 had strong activity in almost all the tissues except the seeds. All these results indicated that this promoter had a bright application prospect in the plant genetic engineering.By analyzing different 5’deletion mutants of the TsVP1 promoter, we found the activity of PT2 was much less than PT1. A 856bp region (-2200 to-1344) was found to contain enhancer element that increased gene expression levels. Two AAATGA motifs, which may be the key elements for the anther specific expression profile, in the deleted TsVP1 promoters (PT2 to PT6) were also identified.A 130bp region (-667 to -538) was finally identified which may be the key sequence for the salt stress response by analyzing the different mutants both with and without salt stress. Agrobacterium-mediated GUS transient assay in tobacco leaves suggested that this 130bp region was sufficient for the salt stress response. Bioinformatic analysis also revealed that there may be novel motifs in this region that were the key elements for the salt stress responsive activity of the TsVP1 promoter. Therefore, further research on this region would be important to reveal the salt stress response mechanism of this promoter.Using this 130bp region as the bait, we identified two proteins which could bind to this region by a yeast one-hybrid system. The two proteins were named as TsNacl and TsVOZ1, respectively.TsNacl has a ORF with 918bp length and share about 86% similarity with RD26 (AT4G27410) in the nucleotide level and 92% similarity in the amino acid level. RT-PCR results indicated that TsNacl was induced by salt, drought and ABA treatment, although there was some differences in the fold change levels. First, its expression in the roots was much less than the leaves under normal condition, but the change folds after stress treatment in the roots was much more than leaves. Second, it was sensitive to ABA treatment, especially in the roots. Its expression could increase to 1000-folds after 12 hours of ABA treatment. In contrast, its expression was not sensitive to drought stress, especially in the leaves. These results showed that TsNacl may be involved in the response to salt stress in Thelhngiella halophila, and ABA was related to this response.The TsNacl protein was expressed in E.coli strain BL21 and was used in the EMSA experiment. The expressed protein could bind to the 130bp region in vitro and this binding was specific. Further study to reveal the relationship of TsNacl and its target genes would be of great importance.According to the EMSA experiment we know that TsNacl protein could bind to the 130bp region. RT-PCR results showed that the expression level of TsNacl could be induced by salt stress. All these results indicated that TsNacl may be the upstream TF we were looking for.In the subsequent experiment, we constructed the over-expression and RNAi constructions of these two genes and transformed both Arabidopsis and Thelhmgiella halophila to study their function in the plant salt tolerance. The increased expression level of TsNacl in Arabidopsis could increase the plant salt-tolerance while the decreased expression level of TsNacl could increase the salt-sensitivity of transgenic plants.Identifying the TsNacl binding sites in TsVP promoter could provide a valuable reference for understanding the plant salt stress response mechanisms, and it may also provide a useful promoter element for the plant genetic engineering. So it was meaningful to identify the TsNacl binding site in the TsVP promoter. Using excessive different non-labeled DNA to compete with the TsNacl protein binding DNA, we identified a 20bp sequence (GAATATACCATGGATAAGCA) which may be the protein binding site. This DNA sequence contains a CATG element. NAC family protein binding site was thought to be CACG, but Trans et al shows that the protein family in Arabidopsis also binds to a MYC-like CATGTG sites. We speculated that CATG may be the core sites for TsNacl protein binding, but the combination of several nucleotides around should also be needed. Further mutations combined with the EMSA experiment to find the specific binding of the protein sites will be important.Based on this analysis, we did some further research on TsNacl. The binding site in the whole genome of Arabidopsis was analyzed by CHIP-on-chip. The results showed that the protein could bind to the promoter region from 284 genes in Arabidopsis. These 284 genes involved in many biological processes, including metabolism, development, localization, stimulus response, reproduction, protein phosphorylation, redox and the regulation of biological processes. It was noteworthy that in these 284 genes, about 70 genes encoded transcription factors, accounting for 25%, while the proposed Arabidopsis transcription factors represent only about 5% of the number of all the genes. This meaned that in the TsNacl target genes, the transcription factors share ratio was around 5 times of the normal ratio, and its target genes included some well studied genes such as the DREB gene that was recognized as stress tolerance-related transcription factors. This also showed that TsNacl genes played an important role in plant stress tolerance. TsNacl could control the expression of functional genes by direct regulation or through some little molecular transporters, structure protein or binding protein. This result indicated this transcription factor may be a key protein in the regulation net and it may have a rather upstream position. In addition, AVP1, the TsVP homologous gene in Arabidopsis was not included in these target genes. According to the previous promoter analysis, we know AVP1 was not induced by salt stress although it was related to stress resistance. The results showed that the TsNacl was not involved in the AVP1 gene regulation, which was consistent with our expectation.In addition, we also identified another protein TsVOZ1 which could bind to the bait plasmid based on the sequence of TsVP promoter in yeast. TsVOZ1 shared about 85% necleotide similarity and 90% amino acid similarity with AVOZ1. Its expression was induced by salt stress, but not by drought or ABA treatment. And the change folds were much less than that of TsNacl. According to the research results of Mitsuda et al., we found a "GCGTCGGCTGCACGC(-274 to-259)" sequence in the TsVP promoter region which may be the binding site of TsVOZl protein. This sequence was not included in the 130bp region we identified before. Changing the expression level of TsVOZl did not change the salt tolerance of transgenic Arabidopsis. This result indicated that TsVOZ1 could bind to the TsVP promoter, but it was not involved in the salt stress response regulation of this gene.In this study we analyzed the function of TsVP promoter and its upstream binding protein. This result would provide new cis-elements and salt-tolerant genes for plant transgenic engineering and also important information for further understanding of the difference in the salt-tolerance machanisms between Arabidopsis thaliana and Thelhungiella halophila.更多还原