【作者】 郑会全；

【导师】 张志毅；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2010， 博士

【摘要】 毛白杨(Populus tomentosa Carr., Chinese white poplar)是我国特有的白杨派(Section Leuce)乡土树种,具有分布广、速生、材质优良及抗逆性强等特点。然而,有关毛白杨抗逆性分子机理研究,尤其在抗病性方面,基础十分薄弱。对毛白杨抗病免疫反应系统中重要基因启动子的结构、功能及作用机制进行深入研究是分析毛白杨抗病免疫性的一个有效切入点,迄今未见任何报道。TIR-NBS类抗病相关基因是调控植物抗病免疫反应的一类重要遗传因子,但对调控该类基因表达的启动子结构、功能及作用机制至今仍不清楚。为此,本文以毛白杨一个典型的TIR-NBS类抗病相关基因(PtDrl02)为切入点,首先对其组织与诱导表达模式进行分析,进而采用Touch-DownPCR技术分离获得PtDrl02基因上游启动子区,在运用生物信息学手段对启动子中包含的顺式调控元件进行功能性预测的基础上,构建了启动子全长及5′缺失序列植物表达载体,开展了烟草与杨树的遗传转化研究,对启动子的组织与诱导表达特性进行了分析。同时,鉴定了启动子关键调控域及重要顺式作用元件。通过构建PtDrl02启动子双向表达植物载体对启动子上游调控元件的方向性进行了初步鉴定。利用瞬时共转化与表达技术,研究了毛白杨PtWRKY1转录因子对PtDrl02启动子转录活性的调控作用。此外,对PtDrl02基因5′UTR序列调控PtDrl02启动子活性的分子机制进行了深入分析。基于上述研究,得到如下主要结果：1.以三倍体毛白杨[(P. tomentosa×P. bolleana)×P. tomentosa]杂种抗病无性系‘L9’为试材,证明了PtDrl02基因在叶片、叶柄及幼茎组织处特异表达,但与内参ACTIN基因相比,表达水平明显较低。通过对杂种毛白杨苗木顶端第2-4叶片间的区域(包括叶片与茎段)进行病原相关诱导处理,进而对PtDrl02基因的表达作qRT-PCR分析发现,机械创伤、MeJA以及SA等病原相关诱导因子均能显著诱导PtDrl02基因的表达；但对不同因子,PtDrl02基因表现出的转录模式不尽相同,如PtDrl02转录子含量在创伤处理6 h后迅速增加,在12 h时达到峰值,表达量是处理前的9.70倍；而用MeJA或SA处理后6 h时,PtDrl02表达量就达到了顶峰,随着处理时间的延长,基因表达量逐渐下降；在SA诱导下,PtDrl02基因表达在12 h和24h时呈相似水平,这与MeJA诱导有所不同。2.首次分离出PtDrl02基因上游启动子区,长度为986-bp。采用PlantCARE、PLACE、NSITE-PL及ScanWM-P等四种生物信息学分析软件分别对启动子区潜在的顺式调控元件进行了预测,发现PtDrl02启动子中不仅存在TATA-box、CAAT-box及GC富集序列等高等植物基本调控元件,同时还包含了多种逆境响应顺式调控元件,如病原(或病原相关)诱导响应元件W-box和GT-1 motif等。3.以GUS基因为报告基因,构建了PtDrl02启动子全长序列植物表达载体并开展转化烟草研究。GUS组织化学染色分析显示,PtDrl02启动子主要在转化植株地上部位器官表达,且集中在叶脉、叶柄和茎的皮层组织以及茎的髓部,具有明显的组织特异性。此外,5′序列缺失分析结果表明,PtDrl02启动子-985／-669和-669／-467序列为决定PtDrl02启动子基本活性的正调控区。4.系统比较了PtDrl02启动子全长与5′缺失序列转化烟草(茎部组织)在病原相关诱导、ABA与NaCl诱导下的GUS报告蛋白活性。结果发现,包含W-box元件的-669／-467与-244／0启动子片段均有效介导了启动子的机械创伤诱导响应过程,其中-669／-467序列同时也是启动子响应MeJA信号诱导的重要区域；具有GT-1 motif的-467／-244启动子片段则是PtDrl02启动子应答SA与NaCl诱导的关键区域,而包含ABRE motif的-985／-669启动子区对PtDrl02启动子ABA诱导反应的产生起决定性作用。另外,通过构建PtDrl02启动子双向表达植物载体,并对它们的表达活性进行分析,首次证明了PtDrl02启动子上游调控元件在调控下游基因表达时,包括在MeJA诱导条件下,不具方向性。研究还发现,毛白杨PtWRKY1转录因子参与了PtDrl02启动子的活性调控过程。PtWRKY1基因的表达抑制了PtDrl02启动子全长序列的活性,而启动子5′缺失序列(-669／0、-467／0和-244／0)活性却能够被PtWRKY1激活,表明PtWRKY1转录因子在调控PtDrl02启动子活性时具有双重功能。5.PtDrl02启动子下游具有一个5′UTR序列,该序列能够对PtDrl02启动子调控下的基因表达起抑制作用,这种抑制作用主要发生在基因转录与蛋白翻译两个水平上。此外,在机械创伤、MeJA、SA、ABA以及NaCl诱导条件下,PtDrl02 5’UTR序列同样对PtDrl02启动子驱动下的基因表达起重要的调控作用。6.采用农杆菌介导的遗传转化法成功获得PtDrl02启动子-GUS (P-985/GUS)转基因毛白杨无性系。在此基础上,对PtDrl02启动子驱动下的GUS基因表达进行分析,结果显示,PtDrl02启动子在转基因毛白杨中表现出原有的组织特性即在地上组织处特异表达,其活性明显受机械创伤、MeJA、SA、ABA以及NaCl诱导。进一步研究还发现,由PtDrl02启动子驱动下的基因(GUS)转录与病程相关蛋白基因(PR-1、PR-5与PR-10)表达在时间模式上呈一定程度的同步性。由此可以推断,PtDrl02启动子能够调控下游抗病基因在转化毛白杨中协同病程相关蛋白基因,进而共同参与植株抗病免疫反应过程。本研究对毛白杨PtDrl02启动子的分子结构与功能进行了分析,初步阐明了该启动子调控基因表达的分子机制,结果为毛白杨抗病免疫调控机理研究提供了一个有效的突破点,同时为植物TIR-NBS类抗病相关基因表达调控机制分析,以及PtDrl02启动子应用于植物尤其是杨树抗病基因工程研究奠定了重要基础。更多还原

【Abstract】 Populus tomentosa Carr. (Chinese white poplar) is a wildspread indigenous tree species of Section Leuce in north China. It has several beneficial traits including rapid growth, qualified wood and strong resistance to multiple biotic and/or abiotic stresses. However, regarding the molecular basis of the stress responses of this species, our current knowledge is rather limited, typically for that on the innate immune system. Thus, further studies are highly required. Because the fine-turn of the innate immunity, to a large extent, was determined by the expression of a set of key R (disease resistance)/R-like genes, the structural and functional dissection of these gene promoters can therefore provide a pioneer cue for the immunity studies of Chinese white poplar. While considering that the molecular mechanism underlying the expression regulation of plant TIR-NBS-encoding disease resistant like genes is still unkonw, in this study, we first conducted an investigation on the functional role of a novel TIR-NBS-encoding disease resistance like gene (PtDrl02) promoter from triploid white poplar [(P. tomentosa×P. bolleana)×P. tomentosa] (in Chinese white poplar). The tissue specific expression pattern of PtDrl02 gene and its defense-related stimulated expression profiles were shown. Following that, the promoter sequence of PtDrl02 gene was obtained using a Touch-Down PCR assay. Potential cis-acting regulatory elements within the PtDrl02 promoter region were then predicted with bioinformatic methods. Based on the construction of the chimeric expression vectors respectively bearing the full-length and 5’progressive deletions of the PtDrl02 promoter, and their genetic transformation on tobacco or poplar plants, as well as following reporter detection assay, the tissue-specific and inducible properties of the PtDrl02 promoter were revealed. Moreover, the functional regulatory segments have also been identified. Additionally, the orientation of the upstream regulatory element of PtDrl02 promoter in directing gene expression unpon the treatment of MeJA or not was verified with two artificial PtDrl02 bidirectional promoter vectors. Our study also suggested that the PtDrl02 gene 5’untranslated region (5’UTR), as well as a Populus WRKY transcription factor, PtWRKY1, was involved in the regulation of PtDrl02 promoter activities. The main results obtained in this study are shown as follows:1. RT-PCR results indicated that the PtDrl02 gene was active in the leaves, petioles and green, young stems of triploid white poplar, while there was no evidence of expression found in bark and roots, suggestive a tissue-specific expression patten of PtDrl02 gene. Notably, transcript abundance of PtDrl02 gene in these tissues was significantly lower than that of the reference ACTIN gene. qRT-PCR was carried out using total RNA extracted from the poplar aerial parts between the second and fourth leaves at different time points after the treatments of wounding, methyl jasmonate (MeJA) or salicylic acid (SA). The PtDrl02 transcript level could be substantially induced by wounding with a maximal level at 12 h. Treatment with MeJA or SA also significantly increased the transcription of PtDrl02 gene, especially at six hours.2. A 986-bp promoter fragment of PtDrl02 gene was obtained by Touch-Down PCR with triploid white poplar genomic DNA as a template. Computer-assisted analysis revealed that the present promoter sequence contained several basal regulatory elements including TATA-box, CAAT-box and GC-rich module, and a cluster of stress-responsive elements such as W-box, GT-1 motif and ABRE motif.3. The full-length PtDrl02 promoter (986-bp) was fused to the GUS reporter gene in a plant expression vector (MfpBI121) and transferred into tobacco plants. The transgenic tobacco lines, designated as P-985, were then subjected to histochemical GUS staining, which clearly revealed the tissue specificity of PtDrl02 promoter. GUS gene expression occurred mainly in the aerial parts of the plants, but was strictly confined to the cortex tissues of leaf veins, petioles, stems, and the stem piths. GUS was not expressed in the mesophyll cells, trichomes, epidermis, and vascular bundles. These data suggested that the PtDrl02 promoter displayed a tissue-specific expression pattern, which differed from that of the cauliflower mosaic virus 35S (CaMV 35S) promoter that served as a positive control, directing constitutive GUS reporter expression in the transgenic tobacco. Additionally, it was found that the PtDrl02 promoter had two positive regulatory regions (-985 to-669, and-669 to-467; the transcription start site taken as+1) that were responsible for its basal activity, as revealed by 5’deletion analysis assay.4. The PtDrl02 promoter activity can be induced by treatment of wounding, MeJA, SA, ABA (abscisic acid) or NaCl in transgenic tobacco stems. Impresively, deletion analysis further revealed that the promoter segments from-669 to-467 and-244 to 0 contained novel cis-elements in response to wounding/MeJA, and wounding alone respectively, while the-467 to-244 fragment was required for SA-and NaCl-inducible expression of the promoter, and additionally it was found that the-985 to-669 sequence was the promoter region essentially for ABA-induced expression. These regulatory regions exhbited a strong correlation with the predicted stress-responsive motif (W-box, GT-1 motif and ABRE motif) locations in promoter, implying that the PtDrl02 promoter functioned by multiple cis-regulatory elements in distinct and complex patterns to regulate PtDrl02 gene expression. Using the Agrobacterium-mediated transient expression approach, we were able to demonstrate that the PtWRKY1 transcription factor was involved in the regulation of the PtDrl02 promoter activity. Full-length promoter expression was suppressed by the PtWRKYl factor, while expression of PtWRKYl gene significantly upregulated the activity of the truncated promoter regions (-669 to 0,-467 to 0, and-244 to 0) of PtDrl02. This result suggested that the PtWRKY1 factor had dual functional activity in regulating the PtDrl02 promoter expression. In the present study, we also found that the upstream regulatory elements of the PtDrl02 promoter could behave in a bidirectional manner in directing gene expression with or without the treatment of MeJA. 5. We have demonstrated that the PtDrl025’UTR sequence conferred a negative effect on the PtDrl02 promoter-directed GUS gene expression at both the transcription level and the translation level in unchallenged transgenic tobacco. Moreover, a complex regulatory effect from the 5’UTR sequence on the PtDrl02 promoter activity under different stimulus-inducible conditions (treated with wounding, MeJA, SA, ABA, or NaCl) was also found.6. To determine the transcriptional activity of the PtDrl02 promoter in transgenic poplar, a total of six PtDrl02 promoter-GUS (P-985/GUS) transgenic P. tomentosa were obtained via an Agrobacterium-mediated transformation method. Histochemical staining, RT-PCR and qRT-PCR results consistently revealed an aerial-specific expression pattern of the PtDrl02 promoter in transgenic P. tomentosa. Furthermore, it was found that the transcriptional activity of the PtDrl02 promoter could be significantly induced by the treatment of wounding, MeJA, SA, ABA, or NaCl but apparently with a time course manner in transgenic P. tomentosa. Our study also suggested that the PtDrl02 promoter directed GUS transcription, to a certain extent, paralleled to that of the endogenous pathogenesis-related genes (PR-1, PR-5 and PR-10) in time, implying a biotechnolgy potential of the PtDrl02 promoter in use for genetic engineering P. tomentosa with disease resistance, assuming that the foreign R gene (s) could be temporally controled by PtDrl02 promoter and co-functioned with PR genes.The aforementioned findings increased our understanding of the molecular structure and function of the PtDrl02 promoter, as well as its regulated role in directing gene expression, which therefore provided new insights into the regulation of the innate immunity of Chinese white poplar. Additionally, these findings also paved a way for further investigation of the regulation of plant TIR-NBS-encoding disease resistance like genes, as well as the biotechnology application of the TIR-NBS-encoding gene promoters, including PtDrl02 promoter, in transgenic plants, typically for that of the Populus spp..更多还原