【作者】 韩胜芳；

【导师】 肖凯； 崔彦宏；

【作者基本信息】 河北农业大学 ， 作物栽培学与耕作学， 2009， 博士

【摘要】 磷素是植物必需的大量无机营养元素,是核酸、磷脂和ATP等生命大分子的重要组分。提高作物对土壤中磷素和施用磷肥的利用效率,对于促进农业的可持续发展具有重要意义。本项研究针对目前有关水稻磷效率生理和分子机制有待深化的现状,以典型粳稻品种为材料,开展了水稻耐低磷能力基因型差异及其生理基础、水稻应答磷胁迫特异表达基因鉴定和介导植株磷素吸收、转运的水稻磷转运蛋白基因(OsPT2和OsPT4)分子特征、表达特性和功能的研究。主要研究结果如下:1.以10个粳稻品种为材料,研究了不同供磷水平下供试品种的生长和磷素吸收特性。结果表明,缺磷(20μM Pi)条件下,供试水稻品种单株磷累积量具有较大差异,可划分为高效、中效和低效三种类型。单株干重和全磷含量与单株磷累积量在缺磷条件下呈显著或极显著正相关。表明缺磷条件下植株生长状况与吸磷能力强弱具有紧密联系。缺磷条件下,供试水稻株高、单株叶面积与单株磷累积量表现趋势相同,但单株根数、平均根长和根系体积与磷素吸收关系不密切。研究表明,磷胁迫条件下根系的磷吸收能力,是影响水稻品种磷吸收量的主要原因。缺磷条件下吸磷量相对增多,是磷高效品种植株性状相对改善的重要生物学基础。2.以磷高效品种TP309和优质8号、中效品种垦优2000和新90-3及低效品种9618和早88-1为材料,研究了缺磷条件下供试品种的光合特性和细胞保护酶活性。缺磷条件下,随植株生长进程,供试品种的光合速率(Pn)均不断下降;但在同一测定时期以磷高效品种最高,中效品种次之,低效品种最低。单株叶面积表现特征与Pn不尽不同。缺磷条件下,叶绿素(Chl)含量、可溶蛋白含量和超氧化物歧化酶(SOD)活性也表现为随品种磷吸收效率提高而增大,丙二醛(MDA)含量则相反。气孔导度(Gs)和蒸腾速率(Tr)则与磷效率不具相关性。Pn、Chl、可溶蛋白含量和SOD活性与单株磷累积量均呈显著或极显著正相关,MDA与单株磷累积量呈显著负相关。表明可用上述生理生化参数作为鉴定水稻耐低磷能力的参考指标。研究表明,较强的SOD活性是高效供试品种在缺磷条件下细胞膜脂过氧化程度低的重要原因,并在维持光合器官功能中具有重要作用。3.采用cDNA-AFLP技术,鉴定了磷高效品种TP309在低磷胁迫下的特异表达基因。共鉴定特异表达基因54个,其中22个具有推定功能。已知功能基因分别归属于信号转导、转录调控、氨基酸合成、物质运输、逆境响应和蛋白质合成等6个功能类别。14-3-3类似蛋白基因(AF451190)在低磷胁迫下增强表达,可能对于增强质膜H+泵活力、进而增强在低磷胁迫下的磷素吸收中具有较重要作用。丝氨酸/苏氨酸激酶基因(AK100849)的表达受到低磷胁迫诱导,可能参与了胞内低磷逆境信号的转导过程。信号肽酶基因(AB066265)可能参与了胞内低磷逆境信号传递体中特定蛋白的剪切、加工,介导转至胞内低磷信号的进一步传递。本研究中,鉴定了bHLH型转录因子基因OsPTF1(AY238991)也呈低磷胁迫诱导表达。表明该转录因子在增强供试品种TP309在低磷胁迫条件下的磷素吸收和利用中具有较重要作用。此外,热激转录因子基因(AK065643)参与了下游低磷应答部分基因的转录调控。HAK8基因(AJ427977)上调表达,表明磷胁迫条件下植株的钾素吸收特征也发生一定改变。本研究鉴定了8个应答低磷逆境响应基因,有关上述基因的功能有待进一步探讨。研究表明,水稻对低磷胁迫的响应,存在着植株对低磷信号转导、特定基因的转录调节和植株在生理生化和形态学上对低磷应答等复杂的生物学过程。4.水稻磷转运蛋白基因OsPT2的编码阅读框为1587 bp,编码528个氨基酸,蛋白分子量57.84 kDa,等电点8.68。编码蛋白含有12个跨膜域。系统进化分析表明,OsPT2与水稻OsPT1、PT1、PTs1和OsPT3,及小麦PT8、大麦PT和玉米PT4高度同源。在正常供磷(CK,2 mM Pi)条件下,OsPT2在根系和叶片中均有表达,但以在叶中的表达水平较高。随着磷水平降低,OsPT2在根中的表达增强,在叶中的表达表现为随外界磷浓度变化保持稳定的组成型特征。在低磷(20μM Pi)条件下,在根中表达随着低磷处理时间延长不断增加。低NH4+对根叶中OsPT2的表达产生负向调控效应,但外界NO3-、K+、Fe2+和Zn2+浓度的变化对OsPT2的表达没有影响。OsPT2启动子中含有许多应答非生物逆境的调控元件,其中应答低磷胁迫逆境的PIBS可能与根系中OsPT2低磷增强表达有关。OsPT2启动子驱动报告基因Gus在不同磷水平下的表达结果,与OsPT2在相应磷水平下的表达特征相一致。对野生型植株(CK)和具有不同OsPT2表达水平的烟草转基因系1、4和6 T2植株研究表明,与CK相比,转基因烟草植株的全磷含量没有差异。但转基因系植株的单株干重、单株磷累积量和光合速率较CK均有不同程度的增加,表现为随植株中OsPT2表达水平提高,上述性状或参数数值不断增大。因此,异源表达OsPT2,具有增强植株在低磷胁迫条件下磷素吸收和改善植株光合碳同化能力的作用。5.水稻磷转运蛋白基因OsPT4的编码阅读框为1617 bp,编码538个氨基酸,蛋白分子量58.83 kDa,等电点8.24。OsPT4含有11个跨膜域,各跨膜域的氨基酸残基数量变化在17~27之间。系统进化分析表明,OsPT4与水稻Pht11-2、PT5、小麦PT1、大麦HvPT4和玉米PT2具有较高的同源性。在不同供磷水平下,OsPT4在植株根系中均不表达,在叶片中的表达表现为随介质中磷水平降低不断增强。在低磷胁迫(20μM Pi)条件下,OsPT4在叶中的表达表现为,随着低磷处理时间延长表达水平呈不断增加趋势。叶中OsPT4对外界NH4+、NO3-、K+、Fe2+和Zn2+浓度的变化均不产生应答。研究表明,在OsPT4启动子中,不存在应答低磷胁迫逆境的重要调控元件PIBS,表明OsPT4在叶片中对低磷胁迫的上调应答,与未知的其他调控元件有关。研究发现,OsPT4启动子驱动报告基因Gus在不同磷水平下的表达结果,与OsPT4在相应磷水平下的表达特征相吻合。高表达OsPT4的转基因烟草植株(转基因系1、5和7),与野生型植株(CK)相比,植株低磷处理后的磷累积量没有差异,全磷含量表现下降趋势。但转基因植株的单株干重和光合速率较对照显著增加。此外,通过单株干重和单株磷累积量的比值获得的磷利用效率表现为,高表达OsPT4的转基因烟草植株较CK显著增加。因此,异源表达水稻磷转运蛋白基因OsPT4,对植株在低磷胁迫条件下的磷素吸收能力没有影响,但具有改善植株体内磷素转运和增强低磷胁迫条件下植株磷利用效率的功能。更多还原

【Abstract】 As one of indispensible macronutrients in plants, phosphorus acts as important components of nucleic acid, phospholipids, and ATP. It plays an important role on improving the utilization efficiency of phosphorus in soil and the fertilizers, by which to promote the sustainable development of agricultural production. In this study, the genetic differences and the corresponding physiological mechanism of low-Pi tolerance, the responsive differential-expressed genes to starvation-Pi, and the molecular characterization, expression patterns, and putative function of phosphate transporter genes OsPT2 and OsPT4 in rice have been explored and analyzed. The results were as follows:1. Using ten rice cultivars (Japonica type) to be the materials, the growth and phosphorus acquisition properties of the cultivars under various Pi levels were studied. It was found that there were dramatic differences on the accumulative P amount per plant among the cultivars under the deficient-Pi (20μM Pi) condition. The cultivars could be grouped in high-, mid- and low- type based on the Pi acquisition capabilities. There were significant and most significant correlations between dry weight per plant, total phosphorus content and accumulative P amount per plant, respectively, suggesting that the plant growth status under deficient-Pi was regulated by the plant Pi acquisition capability at high extent. In the meantime, the plant height, leaf area per plant were also shown to be a similar pattern with the accumulative P amount per plant, whereas the root numbers per plant, average root length, and root volume were not correlated with the Pi acquisition capability. Thus, the uptake amount of phosphorus under deficient-Pi condition was mainly affected by the Pi acquisition capability. The relative more amount of Pi was the important biological basis that the high-efficiency rice cultivar had improved plant growth traits when exposed to low-Pi.2. Using different Pi efficiency cultivars, including high-efficiency cultivars TP309 and Youzhi 8, mid-efficiency cultivars Kenyou 2000 and Xin 90-3, and low-efficiency cultivars 9618 and Zao 88-1, to be the materials, the photosynthetic characterization and the activities of cellular protection enzymes were studied. Under deficient-Pi condition, the photosynthetic rates (Pn) in the tested cultivars were gradually decreased with the growth process. But the Pn were the highest in the high-efficiency cultivars, then in the mid-efficiency cultivars, and were the lowest in the low-efficiency cultivars. The chlorophyll (Chl) concentration, soluble protein content, and superoxide dismutase (SOD) activity showed similar patterns with Pn in the tested cultivars, with higher values in the high-efficiency cultivars. But the malondialdehyde (MDA) content was shown the reverse pattern, with the highest in low-efficiency cultivars, then the mid-efficiency cultivars, and the lowest in the high-efficiency cultivars. There were not correlations between stomatol conductance (Gs) and transpiration rate (Tr) with the phosphorus use efficiency. There were significant positive correlations between Pn, Chl, soluble protein content, and SOD activity with accumulative P amount per plant, and significant negative correlation between MDA content and accumulative P amount per plant, suggesting that the above parameters could be used as the criteria for identification of rice genotypes with high capabilities of low-Pi tolerance. It is found that higher SOD activities play important roles on lessening the cellular over-oxidation degree under deficient-Pi condition, and to be further functional in sustaining relative normal running of the photosynthesis mechanism under low-Pi stress.3. Using cDNA-AFLP approach, the differential expressed genes (DEG) responding to low-Pi stress in cultivar TP309 were analyzed. In total 54 DEG were identified, including 22 with putative functions and 32 with unknown functions. The DEGs could be grouped into six functional classes, such as signal transduction, transcriptional regulation, amino acid synthesis, trafficking, stress responsive, and protein synthesis.The gene of 14-3-3-like protein (AF451190)was up-regulated by low-Pi, implying that it play roles in Pi acquisition by improving the H+ -pump activation. The induced expression of serine/threonine gene (AK100849) was possibly involved in the intracellular low-Pi signal transduction. The signal peptidase gene (AB066265) was presumed to be associated with the cleaving and processing of particular signal components located at the intracellular membrane, by which to mediate the low-Pi signal transduction. In this study, OsPTF1(AY238991), the previously identified Pi-starvation responsive gene, being classified into bHLH type transcription factor, was also shown to be low-Pi induced. Therefore, this gene should play roles on improving the plant Pi acquisition under deficient-Pi condition. We also identified a heat-shock transcription factor gene (AK065643), with the putative functions to be studied, was induced by low-Pi. The up-regulated expression pattern of HAK8 (AJ427977) indicated that the potassium uptake pattern in rice was also modified by low-Pi. In this study, in total 8 genes grouped into stress responsive were identified. But the functions of the genes were to be further explored. Therefore, the response in rice plants to low-Pi were involved in a complicate biological process, such as inter- or intra-cellular signal transduction, the transcriptional regulation of the related signal components genes and downstream genes, and the response at physiological, biochemical, and phenotypic levels in plants.4. The open reading frame of rice phosphate transporter gene OsPT2 was 1587 bp, encoding 528 amino acids. The molecular weight of OsPT2 was 57.84 kDa, having 12 conserved transmembrane domains and with an isoelectric point 8.68. Phylogenetic analysis indicated that OsPT2 had high similarities with phosphate transporter genes of rice OsPT1, PT1, PTs1 and OsPT3,wheat PT8, barley PT and maize PT4. Under normal Pi supply (CK,2 mM Pi) condition, the OsPT2 transcripts were detected in all roots and leaves, with a little higher amount in leaves. With the decrease of Pi, the expression level of OsPT2 in roots was elevated. But the expression level in leaves was not changed. Under low-Pi (20μM Pi) condition, the expression levels in roots were increased with the exposed time extension of low-Pi. A negative regulation effects on OsPT2 were found by NH4+, but no regulation effects on OsPT2 were identified by NO3-, K+, Fe2+ and Zn2+. Based PLACE analysis, some important regulatory elements related to stress responsive had been identified. One of them, named as PIBS and previously identified to be a low-Pi responsive element, was possibly involved in the elevation of the OsPT2 expression under deficient-Pi condition. In the transgenic tobacco plants in which the reporter gene Gus (β- Glucuronidase) was under the control of OsPT2 promoter, gave a similar histochemiscal staining results with the OsPT2 expression patterns in roots and leaves under various Pi-supply conditions.The transgenic plants fused the OsPT2 open reading frame were generated. Compared to the wild type plants (CK), the transgenic plants from the lines with varied OsPT2 expression levels, including line 1, 4 and 6, had not changed on the total phosphorus content. But the plant dry weight, accumulative P amount per plant, and photosynthetic rate (Pn) in transgenic plants were all higher than those in CK, showing a pattern of much more improvement on above parameters, with the increase of the OsPT2 transcripts. Therefore, ectopically expression of OsPT2 had a function on improving the Pi acquisition capability and photosynthesis in plants.5. The open reading frame of rice phosphate transporter gene OsPT4 was 1617 bp, encoding 538 amino acids. The molecular weight of OsPT4 was 58.83 kDa, having 11 conserved transmembrane domains and with an isoelectric point 8.24. Phylogenetic analysis indicated that OsPT4 had high similarities with rice Pht11-2, PT5, barley HvPT4 , wheat PT1, and maize PT2. Under various Pi supply (2 mM ~20μM Pi) condition, no OsPT4 transcripts were detected in roots. The expression levels in leaves were gradually increased with the decrease of Pi-supply amount. Under low-Pi (20μM Pi) condition, the expression levels in leaves were increased with the exposed time extension of low-Pi. No regulation effects on OsPT4 by the supply levels of NH4+, NO3-, K+, Fe2+ and Zn2+ were identified. Based PLACE analysis, some important regulatory elements related to stress responsive had been identified, but the low-Pi responsive element PIBS was not figured out, suggesting other regulatory elements being possibly involved in the OsPT4 responding to the low-Pi cue. Similar to OsPT2 promoter transgenic analysis, the transgenic tobacco plants in which the reporter gene Gus (β- Glucuronidase) was under the control of OsPT4 promoter, also gave a similar histochemiscal staining results with the OsPT4 expression patterns.The transgenic plants fused the OsPT4 open reading frame were generated. Compared to the wild type plants (CK), the transgenic plants from the lines with dramatic higher OsPT4 expression levels, including line 1, 5 and 7, had not changed on the accumulative P amount per plant, and a little lower total phosphorus contents. But the plant dry weight and photosynthetic rate (Pn) in transgenic plants were all higher than those in CK. In the meantime, the transgenic plants were all had higher phosphorus utilization efficiencies, the ratios of dry weight per plant to the accumulative P amount per plant, than CK under the deficient-Pi condition. Therefore, ectopically expression of OsPT4 had no functions on improving the plant Pi acquisition capability, but played roles on the transportation of phosphorus in the plants, by which to play roles on increasing the phosphorus utilization efficiency in plants under low-Pi stress condition.更多还原