【作者】 顾冕；

【导师】 徐国华；

【作者基本信息】 南京农业大学 ， 植物营养学， 2011， 博士

【摘要】 磷（Phosphorus, P）是植物生长发育必需的大量营养元素之一,广泛地参与到植物体内的能量转移、信号转导、光合作用等过程。它还是许多生物大分子如核酸、磷脂和含磷蛋白酶类的重要组成部分。然而,由于P在土壤中容易被固定和沉淀,且植物从土壤中吸收的主要是无机态正磷酸盐（Phosphate, Pi）,故相对于其他营养元素,P在土壤中的移动性和有效性均很低,其也因此常常成为农田及自然生态系统中植物生长的主要限制因子之一。植物在漫长的进化过程中发展出了一套适应缺磷环境的形态变化及生理生化方面的机制,包括根系构型的改变、酸性磷酸酶、RNA酶及有机酸的分泌、与丛枝菌根真菌（AMF, Arbuscular Mycorrhizal Fungi）形成共生体系等等。植物对缺磷环境的这些适应性机制都是由其背后一系列精巧的分子机制作为支撑的。近年来,该研究领域科学家们针对这些分子机制做了大量的研究,初步构建了植物缺磷和菌根共生信号转导途径的分子调控网络。这个网络中的“节点”,即基因之间在不同水平存在着复杂的调控或被调控的关系。一个基因在表达丰度或时间空间上的变化可能会造成整个网络的重新调整。虽然我们对植物缺磷和菌根共生信号转导途径这两个分子调控网络的认识正不断深入,发现了在前者中起中心调控作用并可能连接两个网络的关键调控因子PHR （PHosphate Starvation Response）,但根据近年来该领域的研究进展来看,欲全面揭开其神秘的面纱仍有诸多工作要做。为了对这两个网络进行进一步的补充完善,本研究以鉴定植物中参与到缺磷和菌根共生信号途径中的微小RNA （microRNA, miRNA）和转录因子基因两方面作为切入点,取得了以下的主要结果：1.根据已知植物中所有miRNA的成熟片段序列,与茄科植物烟草的GSS（Genome Survey Sequence）和EST （Expressed Sequence Tag）序列进行比对,继而由生物信息学技术从烟草中预测到分属于84个家族共计276个miRNA基因,并分析了全部276个基因的一系列特征参数。发现烟草miRNA也是以多基因家族的方式出现；其成熟片段和前体结构的长度分别集中在21个碱基和75-114个碱基左右；90%以上niRNA前体的A和U含量之和在50%-70%之间；在不同物种中非常保守的缺磷诱导表达的miR399和miR827在缺磷条件下的烟草中表达也发生上调。2.通过miRNA基因芯片结合实验验证的方法在茄科植物番茄中鉴定出23个保守的]miRNA,发现其中16个受到磷素营养或菌根共生信号或二者共同的调控。证明了miRNA基因表达的变化也是植物缺磷和菌根共生信号转导途径中重要的组成部分,且植物缺磷和菌根共生信号转导途径存在部分重叠。3.采用RT-PCR方法结合生物信息学及组织化学分析研究了水稻中缺磷诱导表达的miRNA基因,osa-miR827a （OsmiR827a）及其靶基因。发现OsmiR827a受缺磷信号专一性诱导表达,且其成熟片段可能作为长距离运输信号分子通过韧皮部从地上部转运到地下部。此外,结果表明虽然miRNA827在不同物种中非常保守,但其对靶基因的调控机制可能在单子叶和双子叶植物物种分化的过程中发生了变异。不同于拟南芥AtmiR827的靶基因AtNLA （Nitrogen Limitation Adaptation;属于SPX家族中的SPX-RING亚家族）,水稻OsmiR827a的靶基因属于SPX家族中的SPX-MFS₁亚家族,且其靶基因有两个,OsSPX7和OsSPX8。虽然二者的氨基酸序列一致性大于80%、结构高度相似且都定位于细胞质膜上,但对缺磷信号的响应却完全相反。4.对OsSXP7和OsSPX8两个基因各两个株系的Tos17转座子插入突变体（osspx7-1、osspx7-2和osspx8-1、osspx8-2）在不同供磷条件下的表型、有效磷含量等生理指标进行测定后发现,osspx7-2在高磷和全铵营养且高磷处理下表现出磷中毒的症状,其叶片中有效磷浓度在两种处理下分别为野生型植株的6.2倍和3.8倍,在低磷条件下其叶片中有效磷浓度与野生型植株相比提高了1倍；对于OsSPX8两个株系的突变体osspx8-1和osspx8-2,只有在高磷和全铵营养且高磷两种条件下,其叶片中的有效磷含量比野生型植株提高40%左右。此外,通过对突变体中磷酸盐转运蛋白（PT, Phosphate Transporter）基因及其它缺磷信号转导途径中基因表达的检测及与其它在正常供磷条件下表现出磷中毒症状的突变体或转基因植株的情况相比较,我们推测OsSXP7和OsSPX8至少部分是通过抑制PT基因表达来实现其在植物体内磷的动态平衡过程中的作用。5.在水稻缺磷信号转导途径中心调控因子基因OsPHR2（PHosphate Starvation Response2）的过量表达植株中对OsmiR827a的表达丰度进行了检测,结果表明OsmiR827a可能是新发现的由OsPHR2直接调控的下游基因。6.通过将番茄中菌根特异表达PT基因LePT4的启动子进行分割和定点突变、融合GUS报告基因继而由根癌农杆菌介导的转基因方法转入烟草中检测分析,证明了缺磷信号转导途径中的关键顺式调控元件P1BS （PHR1Binding Sequence）对茄科植物中菌根诱导或特异表达的PT基因的转录激活是必不可少的。而此转录激活过程不需要调控豆科植物血红蛋白基因在根瘤和菌根组织特异性表达相关的NODCON2GM和参与植物的防御反应的WRKY710S这两个顺式调控元件的参与。7.根据拟南芥和水稻中PHR基因的保守序列设计简并引物,以烟草cDNA为模板进行PCR扩增、测序获得其保守区序列后通过RLM-RACE方法获得了烟草PHR同源基因NtPHR2的cDNA全长序列。氨基酸序列比对和进化树分析结果表明,NtPHR2与拟南芥AtPHR1和水稻OsPHR2一样属于MYB转录因子家族中的MYB-CC （Coiled-Coil）亚家族,且与AtPHRl的亲缘关系最近。8.通过基因枪轰击洋葱表皮、RT-PCR和酵母双杂交系统对与烟草中菌根相关候选转录因子基因NtMYCF1和NtPHR2的亚细胞定位、表达模式及其蛋白水平的相互作用作了分析。结果表明,NtMYCF1和NtPHR2均定位在细胞核；NtMYCFl在根部受菌根诱导表达,NtPHR2在根部和叶片中均为组成型表达；NtMYCF1和NtPHR2在蛋白水平可能不发生互作。更多还原

【Abstract】 Phosphorus （P） is one of the essential macro-nutrients for plant growth and development, and plays important roles in energy transfer, signal transduction, and photosynthesis processes. It is also a structural component of many biologically important macro-molecules, such as nucleic acids, phospholipids and P-containing enzymes. However, since P is readily chelated by cations and precipitated in the soil and the only form of P available for plant uptake is inorganic ortho-phosphate （Pi）, the mobility and availability of P is poor. Plants have evolved a suite of responses to adapt to P-deprived environment, including modification of root architecture, release of acid phosphatase, RNase and organic acids, as well as forming mutualistic symbiotic associations with arbuscular mycorrhizal （AM） fungi.In the past decades, a series of elaborate molecular mechanisms underlying these adaptive responses have been intensively studied, and a molecular regulatory network with regarding to Pi starvation and AM symbiosis has been generated. The nodes in this network, namely the genes involved, are closely related. The alteration in abundance, temporal or spatial expression of one gene may lead to a re-orchestration of the entire network. Although an increasing number of genes involved in Pi starvation and AM symbiosis signaling pathways have been and will be identified in diverse plant species, and the function of the conserved central regulator PHR （PHosphate Starvation Response） that might connect the two signalings have been well elucidated, a lot of work should be done to unravel the complex regulatory mechanism of these signalings. In an attempt to get a better understanding of the Pi starvation and AM symbiosis signaling pathways, we tried to isolate and/or functionally characterize the microRNA （miRNA） and transcription factor encoding genes in the present work. The main results acquired are listed as follows:1. Based on all the mature miRNA sequences in plants, we identified276miRNA genes, which belonging to84families, in tobacco by using its GSS （Genome Survey Sequence） and EST （Expressed Sequence Tag） sequences. We analyzed a series of characteristic parameters of miRNA genes, and found that half of the miRNA families have more than one member; the lengths of the mature miRNAs and precursors are～21and75-114nucleotides, respectively; Ninety percent of the miRNA precursors have a Adnine plus Uridine content ranging from50%-70%; the conserved Pi starvation-induced miR399and miR827are also up-regulated by Pi deprivation.2. Twenty three conserved miRNAs were identified in tomato by miRNA microarray, among which sixteen were responsive to Pi starvation or AM symbiosis or both. The data demonstrated that altered expression of distinct groups of miRNA is an essential component of Pi starvation-induced responses and AM symbiosis, and there are common and specific signalings of the P nutrition and AM symbiosis processes.3. The expression of osa-miR827a （OsmiR827a） as well as its target genes was detected by RT-PCR and histochemical analysis. The results suggest that OsmiR827a is specifically induced by Pi starvation, and its mature fragment might act as a long-distance signal molecule transported from shoot to root through phloem. In addition, we concluded that the mechanism of the negative regulation of miR827on its target（s） might have altered during the divergence of monocots and dicots. Unlike Arabidopsis miR827, which targets AtNLA （Nitrogen Limitation Adaptation; a member of the SPX-RING subfamily）, rice miR827a targets two genes from the SPX-MFS₁subfamily, namely OsSPX7and OsSPX8. Although the amino acid sequence identity of the two plasma membrane located genes is82.35%, and their structures are highly similar, their responses to Pi starvation are opposite.4. The phenotype, Pi concentration and some regular physiological parameters were detected in the Tosl7insertion mutants of OsSPX7and OsSPX8（osspx7-1, osspx7-2and osspx8-1, osspx8-2） under varied Pi supply. Pi toxicity symptoms were observed in osspx7-2under both high Pi （HP） and ammonium nutrition （NH4+） conditions, and the Pi concentration in leaves is6.2and3.8folds as high as that of WT plants, respectively. Under low Pi （LP） condition, the Pi concentration in osspx7-2leaves increased1fold as compared to that in WT. In osspx8-1and osspx8-2, the Pi concentration in leaves showed an increase of40%as compared with that of WT plants under HP and NH4+conditions. Moreover, through detection of the expression of some OsPT （Phosphate Transporter） and Pi responsive genes, as well as comparing with other Pi-overaccumulator, we predicted that OsSPX7and OsSPX8regulate in planta Pi homeostasis partially by repression of OsPT expression. 5. The expression of OsmiR827a was also detected in OsPHR2（PHosphate Starvation Response2） overexpressing plants. The result suggests that OsmiR827a might be a novel target directly regulated by OsPHR2.6. Through succession truncation and targeted point mutation of LePT4promoter, we demonstrated that P1BS （PHR1Binding Sequence） is indispensable for the transcriptional activation of LePT4in response to AM symbiosis. Whereas cis-acting elements, NODCON2GM （responsible for the nodule-and mycorrhiza-specific expression of soybean leghemoglobin lbc3and N23gene） and WRKY710S （responsible for plant defense response）, are not involved.7. Based on the conserved region of Arabidopsis and rice PHR genes, we isolated the full cDNA sequence of the homologous gene in tobacco, NtPHR2, by using RLM-RACE technology. The amino acid sequence alignment and phylogenetic tree analyzes showed that NtPHR2is a novel member of the MYB-CC （Coiled-Coil） transcription factor subfamily, and shares the highest sequence identity with AtPHRl.8. The subcellular localization, expression pattern and the potential protein interaction of NtMYCF1and NtPHR2were analyzed by onion epidermal bombardment experiment, RT-PCR and yeast two hybrid system, respectively. The results suggest that NtMYCF1and NtPHR2localize to the nucleus; the transcript abundance of NtMYCFl in root is increased upon AM symbiosis, while NtPHR2is constitutively expressed in both root and leaf; there might be no interaction between NtMYCF1and NtPHR2protein.更多还原