【作者】 张瑞萍；

【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(P)是植物生长发育最为重要的生命元素之一,广泛参与植物体内的生化合成、能量转移、信号转导等代谢过程。由于化学和微生物的强烈固定,大多数自然土壤中的有效磷很低,限制着植物的生长发育。植物为了适应低磷胁迫已经进化出了包括改变根系形态结构、与土壤中的菌根(Mycorrhiza)真菌形成共生体系、诱导高亲和磷转运蛋白(PT)的表达、改善根际土壤化学性质等形态结构和生理生化上的响应机制。然而,植物能够吸收磷素最重要的是实现根系细胞内磷的跨膜运输。植物根系吸收磷需要从质外体运输到共质体,这是一个从低磷到高磷的跨膜运输过程。运输过程中磷与2-4个质子通过共转运的模式进入到细胞质。细胞质内pH值的降低需要质膜H+-ATPase将过多的H+泵到细胞外以保持细胞间的pH平衡。由此形成的电势差为P素的吸收提供了驱动力。水稻作为一种模式作物,是我国最重要的粮食作物,因此开展水稻根系H+-ATPase基因功能研究及其与磷营养的响应机制研究,对揭示植物高效调控吸收和转运磷营养的机理以及培育磷素高效吸收利用水稻品种具有重要意义。本研究以模式品种日本晴(Oryza sativa cv.Nipponbare L.),水稻质膜质子泵基因OsA8完全敲除的纯合突变体osa8(Tos17插入突变)及其野生型植株(Oryze Sativa ssp. Japonica cv.Hitomebore)为材料,通过RT-PCR、转基因等方法研究了水稻根系质膜H+-ATPase对磷胁迫及菌根调控的响应机制和部分H+-ATPase基因的功能。获得的主要结果如下：1.通过测定不同供磷水平下水稻根系质膜质子泵的活性结果显示,磷胁迫水稻根系质子泵的泵活性高于本身的水解活性,Vmax增加,Km降低。说明是由于质膜H+-ATPase编码的基因在转录水平表达的增强,酶蛋白(翻译水平)增强的综合影响,亦或是翻译后水平的调控。磷胁迫时水稻根系的质膜H+-ATPase活性升高,可能对其根系分泌有机阴离子有一定的作用,以此来活化土壤中的难溶性磷,提高植物对磷的吸收。2.通过生物信息学分析和转基因技术分析讨论了水稻质膜H+-ATPase家族中各个基因的特点：水稻H+-ATPase家族基因分布在五个亚家族中(Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ),同家族基因同源性达到80%以上。通过构建启动子与GUS报告基因融合的表达载体,检测了OsA2、OsA3、OsA4、OsA7和OsA8基因的时空表达特征。GUS染色发现,在正常生长条件下OsA2、OsA3、OsA4和OsA7在根尖、侧根发生区、根茎结合部位以及叶片中均有表达,而OsA8基因在这些部位中均不表达或表达很弱。RT-PCR的结果显示,属于质膜H+-ATPase家族基因Ⅰ和Ⅱ两个亚家族的OsA2、OsA3和OsA7基因在植株根系和地上部有较强烈的表达,与GUS染色结果相一致。而没有表达或表达极低的OsA8基因可能在特定的部位,或特殊的条件或时间下表达。3.OsA8启动子融合GUS报告基因的转基因水稻侵染试验结果显示,OsA8基因在菌根水稻根系的丛枝部位不表达。4.osa8突变体及其野生型水稻低磷条件下接种菌根菌(G. intraradices)的砂培试验结果表明,菌根诱导水稻质子泵基因OsA8在根系中的增强表达高达105倍。而同家族的OsA1、OsA2、OsA3、OsA7和OsA9则在根系中出现了显著的下调趋势。与野生型水稻相比,OsA8基因的突变导致了水稻菌根真菌的侵染效率降低了约20%,并且水稻根系中磷浓度的升高、地上部磷浓度的降低。这可能是由于OsA8基因的缺失,降低了水稻中磷素从根系向地上部的转运,使得根系中磷素积累浓度相对较高。从而直接导致了水稻高亲和磷酸盐转运蛋白Pht1家族基因中除OsPT8外的很多成员在根系中表达量的降低。5.OsA8基因超表达水稻对磷营养的响应试验结果显示,OsA8基因超表达提高了水稻质膜H+-ATPase的活性。在低磷胁迫条件下,OsA8基因超表达显著提高了水稻对磷的吸收速率以及植株体内磷素从根系向地上部的转运速率。这与osa8突变体中降低了磷素从根系向地上部的转运结果相一致。这种改变伴随着水稻H+-ATPase基因和水稻高亲和磷酸盐转运蛋白家族基因在转录水平上表达的变化。超表达OsA8基因下调了OsA1、OsA3和OsA9基因在叶片中的表达量。对Pht1家族而言,除了与低磷诱导增强表达的关键基因OsPT2和OsPT6有着密切的关系以外,超表达OsA8基因同时也下调了同家族的OsPT1、OsPT3、OsPT4和OsPT9的表达。总之,通过对敲除质膜质子泵基因的突变体和超表达突变体的分析,我们发现低磷胁迫时,质膜质子泵OsA8基因通过影响根系质膜质子泵的活性,改变了水稻对菌根菌的侵染和磷素的吸收能力,又因下调部分水稻高亲和磷酸盐转运蛋白基因的表达而影响了水稻植株体内根系和地上部磷的分布情况,进而反馈调控了Pht1家族其他基因的表达特征。由此,OsA8基因在响应低磷胁迫和菌根菌侵染时表现出来的对磷素吸收和磷素从根系向地上部转运速率的重要作用,有望应用于通过转基因手段培育适应低磷条件生长和促进菌根形成的磷素高效利用的水稻新品种研究。更多还原

【Abstract】 Phosphorus (P) is essential for plant growth and development due to its involvement in the processes of energy metabolism and synthesis of nucleic acids and membranes. However, the low availability of soil P is a major constraint for crop production in many agricultural systems worldwide. Higher plants thus alter their architecture and metabolism to acquire sparingly soluble P from soil. It was reported that plants are able to mobilize P by acidification of the rhizosphere by release of H+ from the roots to balance excess intake of cations over anions. Pi uptake into the root symplasm involves transport from the apoplast where Pi concentration is less than 2μM, across the plasma membrane (PM), and to the cytosol where Pi concentration ranges from 5-17 mM. The negative membrane potential and the large difference between external and internal Pi concentration necessitate that a steep electrochemical gradient must be overcomed for Pi transport into root cells, thus a high-affinity, energy-consuming transport mechanism driven by PM H+-ATPase is required. The mechanism underlying H+ release is a primary process caused by H+-ATPase activity in the plasmalemma of root cells. This enzyme acts as a primary transporter by pumping protons out of the cell, thereby creating pH and electric potential differences across the PM. Rice (Oryza sativa), an economically important crop and a monocot model plant for scientific research, possesses ten PM H+-ATPase genes. Consequently, research on functional characterization of rice PM H+-ATPase genes with regard to rice nutrients uptake and translocation is of extreme importance for understanding of the mechanism and germplasm enhancement.The studies on the adaptation of plant PM H+-ATPases to Pi deficiency were all carried out by dicotyledonous plants, while little is known about that of the monocotyledonous plants. In the present work, the adaptation of rice root PM H+-ATPase activity to Pi deficiency was studied. Rice plants (Oryza sativa cv. Nipponbare L.) were fed with or without phosphate in hydroponics culture experiment. At the seedling stage the PM of rice roots was isolated by two phase system. The PM H+-ATPase hydrolytic activity was analyzed for elucidating the response of the PM H+-ATPase of rice root to Pi deficiency. Moreover, tissue localization analysis for rice H+-ATPase genes was performed using transgenic plants carrying promoter::GUS (β-Glucuronidase) reporter fusions. Furthermore, with wild type (WT) rice (Oryze Sativa ssp. Japonica cv. Hitomebore) as control, we investigated the physiological function of OsA8, a member of rice PM H+-ATPase gene family, by comparison of the responses between the Tos17 insertional homozygous mutant of OsA8 gene and its WT in nutrient uptake and translocation under arbuscular mycorrhizal (AM) colonization. The main results are shown as follow:1. The adaptation of PM H+-ATPase of rice roots to Pi deficiency is supported by the following results obtained in vitro. Compared with roots from Pi-sufficient plants, the Pi-deficient rice roots had higher hydrolytic and pumping activity of PM H+-ATPase as well as Vmax. The higher activity of H+-ATPase of rice roots under Pi deficiency was mirrored either by a higher expression rate (transcriptional level) of genes encoding PM H+-ATPase or an enhanced synthesis rate of the enzyme proteins. Higher H+ pumping activity than hydrolytic activity, and a higher Vmax, with a lower Km of PM H+-ATPase data suggest that the enhancement of PM H+-ATPase activity by Pi deficiency in rice roots could also be regulated at post-translational level.2. Multiple sequence alignment and phylogenetic tree analysis showed that the ten rice H+-ATPase genes, which could be grouped into five subfamilies, are very closely related with sequence identities over 80%. This indicates that these genes might have evolved through gene duplication events during rice evolution.Transgenic rice plants carrying promoter::GUS fusions were used for the determination of spatial expression pattern and promoter strength of OsATPase genes (OsAs). The expression of OsA2, OsA3, OsA4 and OsA7 was detected in the root tips, lateral roots, root-shoot junction and leaves under normal conditions. While the expression of OsA8 was barely detectable under normal conditions. A possible explanation for this could be that its expression is restricted to particular cell types.3. In contrast to histochemical analysis of GUS activity driven by other promoter fragments of AM-induced genes of solanaceous species or rice. We did not observe any reporter gene activity in any tissues with the OsA8 promoter under AM colonization.4. OsA8 knockout mutants showed a lower colonization rate and higher Pi concentration in roots compared with WT plants. Interestingly, expression of genes of H+-ATPase family other than OsA8 and genes belonging to the Phtl family showed similar expression patterns, namely mycorrhizal symbiosis decreased of the OsAl, OsA2, OsA3, OsA7, OsA9 and Phtl family gene (except OsPT8) expression in osa8 mutant root.5. Two independent transgenic lines with enhanced OsA8 expression showed increased activity of PM H+-ATPase under normal condition. Pi uptake and particularly the translocation of P from roots to shoots were also enhanced. These results are consistent with the enhanced expression of genes belonging to PM H+-ATPase and Phtl family. OsA8 over-expression decreased OsAl, OsA3 and OsA9 of H+-ATPase family and OsPTl, OsPT3, OsPT4 and OsPT9 of Phtl family.The results indicated that OsA8 played very important roles in the inoculation of rice roots with AMF and uptake and translocation of Pi, as well as in regulation of root growth in the rice. Under Pi deficieny and AMF inoculation, Pi uptake and translocation was changed by OsA8 gene affected PM H+-ATPase activityExpression of many other Pi transporters was regulated by changed Pi concentration in the OsA8 gene knockout or over-expression rice roots and shoots. It was concluded that maintenance of OsA8 gene’s expression is critical to keep normal AMF inoculation and Pi uptake and translocation in addition to normal root morphology of rice plants.更多还原