【作者】 丁明全；

【导师】 陈少良； 沈昕；

【作者基本信息】 北京林业大学 ， 植物学， 2011， 博士

【摘要】 胡杨为我国西北干旱盐碱区域唯一能够成林的高大乔木树种,在生态脆弱地区的生态稳定性方面具有不可替代的作用。近年来,国内外学者从生理生化层面对胡杨的耐盐机制进行了系统研究,但是胡杨耐盐分子机理方面的研究还不系统和深入。据此,我们以胡杨以及盐敏感的群众杨为材料,对NaCl长期和短期胁迫下叶片的表达谱进行了动态分析,拟从基因表达的角度揭示胡杨的耐盐机理。主要研究结果和结论如下：1.利用改良的热硼砂盐法提取胡杨与群众杨叶片的总RNA,该方法能有效的抑制植物体内多酚和多糖的干扰,后续的实验也表明,所得到的高质量RNA能应用于生物芯片的研究。2.利用Affymetrix芯片技术研究了胡杨和盐敏感杨树群众杨的表达谱,发现两种杨树的表达谱存在明显差异,这些差异基本上可以分成两类：非诱导型差异(树种间的表达谱差异)和诱导型差异(盐处理前后表达谱的差异)。对比分析两种杨树诱导型与非诱导型差异表达的基因,就可以推断胡杨耐盐分子机制的有关信息。在非胁迫条件下,许多与抗逆相关蛋白的基因在胡杨中的表达量明显高于群众杨。这些基因包括转录调节因子蛋白基因、离子与多糖转运蛋白基因、细胞信号转导蛋白基因、植物抗氧化胁迫蛋白基因等。这表明,生长在干旱盐碱沙漠地带的胡杨已经通过长期进化形成了特有的耐盐机制。3.胡杨与群众杨的表达谱还存在诱导型差异：在胡杨中,短期盐处理共获得了个467差异基因探针,其中368个基因探针上调,99个基因探针下调；长期盐处理的胡杨共获得52个差异表达基因探针,其中上调43个,下调9个。在群众杨中,短期盐处理共获得458个差异表达基因探针,其中420个上调,38个下调,长期盐处理的群众杨共获得3908个差异基因表达探针,其中上调2047个,下调1861个。在这些差异表达的基因中,我们发现一些基因的表达应与胡杨的耐盐性相关,这些基因包括：(1)转录调节物质类基因：转录调节因子(锌指蛋白,wrky51,MYB11,ABF2)、磷脂酶C、酪氨酸磷酸酶、14-3-3等基因；(2)叶绿体叶绿素合成类基因；(3)糖代谢(糖酵解,三羧酸循环,磷酸戊糖途径)、淀粉合成基因；(4)次生代谢物质(渗透调节物质)的合成：类黄酮类、木质素、甘露糖合成基因；(5)活性氧代谢基因：POD、SOD、谷胱甘肽-S-转移酶、抗坏血酸还原酶、抗坏血酸过氧化物酶、过氧化氢裂解酶、铁(硫)氧还蛋白等；(6)保护蛋白基因：热激蛋白、LEA蛋白、细胞色素P450；(7)各种转运体基因：氮转运体、阳离子转运体、山梨糖类转运体、糖类转运体等。在短期或长期盐处理下,上述这些基因在胡杨叶片中上调表达,但在群众杨中的表达量下降或不变。因此,我们认为这些基因的表达应与胡杨的耐盐性相关。此外,我们还在两种杨树中发现了盐诱导共表达的基因。在短期盐处理下,胡杨与群众杨有40个基因表达探针共上调,我们对这些基因进行了启动子的生物信息学分析,发现这些基因的启动子都具有共同的顺式作用元件的结构,主要响应渗透胁迫。4.在对杨树表达谱分析的基础上,我们研究了重要抗盐基因在两种杨树中的表达特点,发现离子平衡调控、活性氧平衡的调控、光合作用相关基因的表达与胡杨的耐盐性密切相关。(1)在离子平衡调控方面,参与离子平衡调控的Na+/H+逆向转运蛋白(如SOS1、NHD2)和质子泵(如质膜H+-ATPase)等重要蛋白的基因在胡杨中的表达量明显高于群众杨,并且,这些基因的表达量没有受到短期和长期盐胁迫的影响。叶片中离子关系的研究结果表明,在长期盐处理下,胡杨叶片中的Na+离子含量显著小于群众杨。表达谱数据显示,胡杨能够保持Na+/H+逆向转运蛋白以及质子泵基因的高表达,有助于将Na+离子排到细胞外空间。(2)在光合作用调控方面,短期盐胁迫下胡杨上调了包括叶绿素a/b结合蛋白、PSB-Q(光系统亚基Q蛋白)、叶绿体内膜蛋白以及光合作用电子传递链类基因,并且,在长期盐胁迫下,这些基因在胡杨中表达量未有下降,但在群众杨中却明显下调。胡杨能够上调光合作用相关基因,有利于胡杨在盐处理条件下维持光合作用的进行,这与本实验室前期胡杨光合作用方面的研究结果相吻合。(3)在活性氧平衡调控方面,短期盐胁迫下,胡杨能够迅速上调许多抗氧化物酶基因,包括Cu-Zn SOD、CAT、PODs、PRX、TRXs、GRX和GSTs。然而,短期盐处理的群众杨只能上调如APX、阴离子POD、TRX、GRX等少数抗氧化物酶基因。长期盐胁迫下,胡杨的抗氧化物酶基因的表达并没有受到太大的影响。与胡杨不同,在长期盐胁迫下,群众杨叶片中许多抗氧化酶基因的表达发生了很大改变：群众杨在下调如Cu-Zn SOD、H2O2水解酶、APX、PRX以及一止匕POD、TRX、GRX和GST家族成员基因表达的同时,也上调了CAT以及POD、TRX、GRX以及GST另外一些家族成员的基因表达。实验结果说明,群众杨抗氧化物酶基因家族的不同成员对于盐胁迫的响应具有明显差异。胡杨响应盐胁迫上调抗氧化酶基因的表达,有利于胡杨细胞对ROS平衡的调控。Confocal的实验结果显示,在长期盐胁迫条件下,胡杨能够控制叶片H202的水平,避免盐诱导氧化伤害。与胡杨不同,群众杨在长期盐胁迫下,叶片发生H202爆发。这是由于群众杨在初始盐胁迫不能迅速上调抗氧化酶基因的表达,难以控制ROS水平,在长期盐处理后导致ROS爆发,从而诱发氧化伤害。(4)应用了荧光定量PCR技术对代表性的差异基因(包括HAK1、ATGPX2、APX、POD和K+通道蛋白基因)进行了验证,验证结果显示,荧光定量PCR结果和表达谱结果所得的差异基因表达趋势一致,表明表达谱数据真实可靠,数据质量满足数据挖掘的质量要求。5.质膜H+-ATPase在胡杨离子平衡调控和胁迫信号转导方面起到非常重要的作用。表达谱的数据显示,胡杨在非盐胁迫下就能维持H+-ATPase基因(AHA)的高表达,然而,胡杨质膜H+-ATPase的分子调控机制鲜有研究。通过比较不同耐盐性杨树H+-ATPase启动子区域的顺式作用元件,能够了解H+-ATPase基因表达调控的分子机制。本文克隆了胡杨与不耐盐杨树灰杨(AHA)的启动子区域,并构建载体进行了瞬时转化的研究。生物信息学的分析表明,胡杨AHA的启动子结构与灰杨明显不同,胡杨AHA的启动子区域富集了响应ABA的顺式作用元件。瞬时表达的结果表明,胡杨AHA的启动子响应ABA,而灰杨该基因的启动子不具ABA响应能力。总之,胡杨与盐敏感杨树在基因表达方面存在明显差异,在胁迫和非胁迫条件下,胡杨能维持离子转运相关蛋白基因的高表达,保持叶片的K+/Na+平衡,避免Na+离子过度累积对叶细胞的毒害作用。胡杨能迅速上调ROS代谢相关蛋白基因的表达,有助于维持膜系统的稳定性,避免膜系统的氧化损伤。此外,胡杨还能迅速上调光合作用相关蛋白基因的表达,从而有利于胡杨在盐胁迫下固定CO2,合成碳水化合物,为胡杨维持离子平衡和活性氧平衡提供重要的能量来源。更多还原

【Abstract】 P. euphratica Oliv. is a valuable tree species used for afforestation on saline and alkaline desert sits in the north-west China, and plays a crucial role to maintain ecological stability in ecologically fragile areas. Recently, the physiological and biochemical mechanisms of the salt tolerance have been extensively investigated in P. euphratica, however, the molecular mechanism of salt tolerance in P. euphratica is not systematicly and thoroughly investigated. Using the Affymetrix poplar genome array, we explored the leaf transcriptome of salt-tolerant Populus euphratica Oliv. and salt-sensitive P. popularis 35-44 (P. popularis) under short term and long term NaCl stress. The aim is to establish a correlation between the gene expression and molecular mechanism of salt tolerance in P. euphratica.1. A modified sodium borate method was adopted to extract the total leaf RNA of P. popularis and P. popularis. This method was effectively to reduce the interference of polyphenolics and polysaccharides during RNA isolation and purification. The follow-up experiments also confirmed that high quality of total RNA could be successfully used in our microarray analysis.2. The transcriptomes of P. euphratica and P. popularis were compared using Affymetrix microarray systems. We found that there were species difference in the expression profiles between the two poplars, which can basically be divided into two categories:natural (non-induced) differences (differences in expression patterns between the poplar species under non-stress conditions) and salt-induced differences (differences in expression profiles before and after salt treatments). The molecular mechanism of salt-tolerant P. euphratica can be inferred according to the differential expressed genes-natural and stress-inducible genes between the salt-tolerant and sensitive poplar. Under non-saline conditions, the transcription abundance of many genes related to stress resistance in P. euphratica is significantly higher than that in P. popularis. They are genes of the transcription factor protein, ion and polysaccharide transporter, signal transduction protein, and anti-oxidative defence components. The data suggest that P. euphratica plants have evolved relevant mechanisms to tolerate saline environments after a long time adaptation to arid and saline desert.3. There are still salt inducible differences in gene expression profiles between P. euphratica and P. popularis. In ST (short-term NaCl)-treated P. euphratica,368 probesets among the 467 differentially expressed genes were up-regulated while the other 99 probesets were down-regulated. Under LT (long-term NaCl) stress conditions,52 probesets were differentially expressed, among which 43 probesets were up-regulated and 9 probesets were down-regulated. For the salt sensitive species, P. popularis, the ST treatment changed the expression of 458 probesets:420 probesets were increased while 38 probesets decreased. The transcription of 3908 probesets were alterd by the LT stress, among which 2047 probesets were up-regulated and 1861 probesets down-regulated. In these differentially expressed genes, we found that some genes were highly associated with the salt tolerance in P. euphratica. These genes are (1) transcriptional regulators genes such as transcription factors (zinc finger protein, wrky51, MYB11, ABF2), phospholipase C, tyrosine phosphatase,14-3-3 and other genes; (2) chloroplast chlorophyll biosynthesis genes; (3) carbohydrate metabolic genes which are involved in glycolysis, Krebs cycle, pentose phosphate and starch synthesis pathway; (4) secondary metabolite (especially osmotic adjustment) synthesis genes like flavonoids, lignin and mannose synthesis genes; (5) ROS metabolism genes like POD, SOD, glutathione-S-transferase, ascorbate reductase, ascorbate peroxidase, hydrogen peroxide lyase and TRX; (6) protective protein genes like heat shock proteins, LEA proteins, cytochrome P450; (7) various transporter genes:nitrogen transporter, cation transporter, sugar transporter, etc. Noteworthy is that these genes were up-regulated in P. euphratica during short-term or long-term salt treatments, but their expression were decreased or remained unchanged in salinised P. popularis. Therefore, we conclude that the salt inducible genes expression is presumably associated with salt tolerance in P. euphratica. Besides the differentially expressed genes that mentioned above, we also found co-upregulated genes in salt-treated plants of the two species. Under ST treatments, transcription of 40 probesets was enhanced in salinied P. euphratica and P. popularis. We extracted the promoter regions of their homolog genes from P. trichocarpa. The bioinformatics analysis show that the promoter of these genes share common cis-acting elements that are responsible for osmotic stress sensing.4. On the basis of poplar transcriptome analysis, we characterized the expression of some important salt-resistant genes in the two poplar species. We found that the gene transcription of ion homeostasis, ROS homeostasis and photosynthesis is closely related to the salt tolerance of P. euphratica.(1) Genes related to ionic homeostasis. We found that genes like Na+/H+ antiporter such as SOS1 and NHD2 and proton pump genes such as PM H+-ATPase were significantly higher expressed in P. euphratica compared to P. popularis. Moreover, their expression was not decreased by the ST and LT stress in P. euphratica. Na+ content in P. euphratica leaves was significantly less than P. popularis under LT treatments. Our microarray data show that P. euphratica maintained high expression of Na+/H+ antiporter and proton pumpss, which are favorable to extrude Na+ to the extracellular space.(2) Genes related to photosynthesis. In P. euphratica, photosynthesis related genes like chlorophyll a/b binding protein, PSB-Q, the chloroplast membrane proteins and electron transport chain genes were up-regulated during ST treatment, and their expression was not down-regulated by the LT treatments. In contrast, the expression of these genes in P. popularis was decreased by LT stress. Therefore, P. euphratica up-regulated photosynthesis related genes to cope with the salt stress, which is beneficial to maintain leaf photosynthesis under saline conditions. The result is consistent to our previous findings in photosynthesis studies.(3) Genes related to ROS homeostasis. After exposure to ST salinity, P. euphratica showed strikingly up-regulated transcription of a variety of anti-oxidant enzymes, including Cu-Zn SOD, CAT, PODs, PRX, TRXs, GRX and GSTs. Compared with P. euphratica, fewer antioxidant enzymes (e.g., APX, anionic POD, TRXs and GRX) were up-regulated in ST-stressed P. popularis. Unlike ST-stressed plants, expression of antioxidant enzymes in P. euphratica was not significantly altered by LT stress. In contrast to P. euphratica, LT salinity significantly affected the transcription of a large number of antioxidant enzymes in P. popularis leaves. NaCl decreased transcription of Cu-Zn SODs, H2O2 lyase, APX, PRXs and some members of POD, TRX, GRX and GST, but enhanced expression of CAT and several members of POD, TRX, GRX and GST. These results indicate the member-specific response to salinity in each family of antioxidant enzymes in P. popularis. P. euphratica rapidly up-regulated expression of antioxidant enzymes upon salt stress, contributing to the control of ROS production and oxidative damage. Confocal analysis of leaves show that P. euphratica strictly limited the level of H2O2 during the LT stress, thus avoiding the salt-induced oxidative damage. In contrast to to P. euphratica, H2O2 burst occurred in LT-stressed P. popularis leaves. This is due to its inability to increase the expression of antioxidant enzymes at the beginning of salt stress, which resulted in an overproduction of ROS in leaf cells, leading to an oxidative damage over a prolonged period of salinity.(4) We used quantitative RT-PCR to evaluate our microarray data. Several representative genes, e.g., HAK1, ATGPX2, APX, POD and K+ channel gene, were verified. Real-time PCR confirmation showed a consistent tendency of gene expression to the microarray data. This indicates that the data derived from Affymetrix poplar genome array was solid and reliable, therefore, the data is sufficiently qualified to continue the subsequent data mining.5. The plasma membrane H+-ATPase plays an important role in ion homeostasis and stress signal transduction in P. euphratica. Our microarray data show that P. euphratica retained a high transcription abundance of the H+-ATPase gene (AHA) under non saline conditions. However, the regulation of H+-ATPase at molecular levels is unkown. We are able to identify different cis-elements related to stress in poplars by comparing the promoter regions of AHAs in the salt tolerant and sensitive poplar species., This can also help us to understand the expression regulation of AHAs upon the salt treatment. The promoter regions of AHAs were cloned from P. euphratica and a salt sensitive species P. canescens, followed by bioinformatics and transient transformation analyses. Bioinformatics analysis show that the promoter structure of PeAHA is rather different to that of PcAHA. Compared to PcAHA, PeAHA is enriched with cis-elements in response to ABA in the promoter region. Moreover, the transient expression results also confirmed that the promoter of PeAHA could response to ABA treatments, but there was no corresponding response in PcAHA promoter.In conclusion, there were marked differences of gene expression between P. euphratica and salt sensitive species. P. euphratica maintained high expression of ion transporters and channels under normal and stressed conditions, which is helpful for the salt-resistant species to control K+/Na+ homeostasis in leaves. This is a mechanism to avoid excessive accumulation of Na+, and reduces its toxicity in leaf cells. P. euphratica can also rapidly increase the expression of ROS metabolism genes to pre(?)ent oxidative damage. This is able to maintain the stability of membrane system and reduces the membrane peroxidation. In addition, P. euphratica is capable of increasing the expression of photosynthesis-related genes, thus contributing to CO2 assimilation under salt stress. The carbohydrates provide an important source of energy for maintaining ionic homeostasis and ROS homeostasis.更多还原