【作者】 武兆云；

【导师】 邢邯；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2011， 博士

【摘要】 磷是植物生长和新陈代谢所需一种最重要大量元素之一,同时也是核酸、磷脂、ATP、酶、辅酶的一种关键性成分。磷(植物主要吸收磷酸盐形式的磷)缺乏是限制植物生长的第二位的大量元素,其在土壤中以复杂的、难溶的、无机和有机形式存在,不能被植物直接吸收。植物的正常生长和产量依赖于可利用性Pi (Inorganic Phosphate,磷酸盐),然而在很多自然和农业生态系统中植物经常会出现有效和可溶性磷极度缺乏的情况。为了适应磷缺乏,植物已进化获得精巧的调控机制来保持体内磷的动态平衡,包括侧根和根毛的发育、根系构型改变(例如蛋白样和胡萝卜状根的形成)、从根系分泌磷酸酶和有机酸、高低亲和性磷酸盐转运体(phosphate transporter, PT)的诱导。同时很多植物与菌根真菌形成共生关系以便获得更多的Pi。在植物中存在着很多的适应性机制以减轻或应对磷缺乏。这些机制的实现是通过几百个基因表达谱的变化来实现的。这-些感知Pi状态和调整适应机制的调节基因组成网络现在逐渐清晰。目前已鉴定的该网络组成包括转录因子、SPX (Sygl, Pho81,XPRl)亚家族蛋白、非编码RNA和蛋白修饰物(包括参与SUMO化、磷酸化、去磷酸化以及蛋白运输的各种蛋白)。本研究克隆了PT、PHR (phosphate starvation response)、SPX和Gm4等相关基因；并利用生物信息学方法研究了GmPT1和GmPT2蛋白结构；采用绿色荧光蛋白融合PT来研究GmPT1和GmPT2的亚细胞定位；在酵母突变体中研究GmPT1和GmPT2功能和生化特征；采用Real-time RT-PCR研究GmPT1合GmPT2表达模式；利用酵母单杂交的方法研究GmPHR1和GmSPX1的互作；同时,利用主成分分析和隶属函数对20种大豆基因型进行了耐低磷性状的评价。通过研究获得以下研究结果：1.根据主成分和隶属函数分析,评价供试大豆基因型耐低磷性状强弱顺序。供试基因型耐低磷由强到弱的顺序为：赶泰、五河齐黄豆、7650、湘豆4号、先进2号、苏88M-21、Peking、高作选1号、科丰一号、新沂小黑豆、南农1138-2、94-156、87-23、菏84-5、波高、RN-9.通山薄皮甲、皖82-178、湘秋豆2号、垫江早黄豆。2.克隆得到两个PT基因分别在大豆染色体Gm10(41,391,168～41,393,008)和Gm20(42,980,124～42,981,928)上,分别命名为GmPT1(登录号：HQ392508)、GmPT2(登录号：HQ392509)。GmPT1含有1841个碱基其开放阅读框编码536个氨基酸(分子量为58730.46Da)。GmPT2含有1802个碱基其开放阅读框编码536个氨基酸(分子量为58627.29Da)。GmPT1和GmPT2的开放阅读框都是从第23-1633bp,在核苷酸序列上有88.7%一致性,在氨基酸序列上有97.9%的一致性。GmPT1和GmPT2氨基酸序列与拟南芥、番茄、马铃薯和蒺藜苜蓿有着很高相似性。GmPT1和GmPT2与来自真菌的PT——菌根真菌(GvPT)以及酿酒酵母(PHO84)有着很高的一致性。GmPT1氨基酸序列与GvPT (登录号：Q00908)、PHO84(登录号：P25297)分别有76%、61%一致性；GmPT2氨基酸序列与GvPT(登录号：Q00908)、PHO84(登录号：P25297)分别有76%、63%一致性。GmPT1和GmPT2(?)勺疏水性分析表明这两个转运体均有12个跨膜区域(TM, transmembrane),这是PT的共同特征,与PT的亲和性无关。GmPT1和GmPT2(?)勺生物信息学预测结果表明这两个蛋白的二级结构含有12个跨膜区域,在TM6和TM7间存在一个大亲水环。3.利用Tbpred预测服务器来预测GmPT1和GmPT2亚细胞定位,其预测结果为GmPT1和GmPT2是整合性膜蛋白。为了验证GmPT1和GmPT2亚细胞定位,将绿色荧光蛋白(GFP)融合到GmPT1或GmPT2的ORF的3’端。在以GmPT1/GFP或GmPT2/GFP进行转化的细胞可以观察到细胞周围有着清晰的荧光信号,转化空载的细胞则可以观察到整个细胞充满荧光信号。GmPT1/GFP和GmPT2/GFP融合蛋白定位在细胞的周边,表明这两个蛋白定位在细胞膜。这个结果与早期生化研究一致,这些数据表明GmPT1和GmPT2定位在细胞膜。4.利用抑制剂来验证Pi运输的pH依赖性。Pi运输活性在pH值4-7范围内进行研究,其活性在不同的pH值下表现出差异——当pH值为4时其活性达到最高并随着pH值升高其活性下降。为了研究质子驱动力对Pi运输活性的影响,本实验使用解偶联剂——2,4-二硝基苯酚(DNP)和羰基羟基氰氯苯腙(CCCP),这些解偶联剂可以破坏细胞膜内外的质子梯度。DNP在浓度为100gM时Pi吸收率相比较无抑制剂的对照减至79%(GmPT1)和82%(GmPT2)。CCCP在浓度为100μM时Pi吸收率相比较无抑制剂的对照减至77%(GmPT1)和80%(GmPT2)。钒酸盐——一种P-类型H+-ATPase(?)(?)制剂——在浓度为100μM时Pi吸收率相比较无抑制剂的对照减至82%(GmPT1)和83%(GmPT2)。这些结果表明其吸收率相比较对照明显地降低了。这些结果证实了GmPT1和GmPT2(?)(?)Pi/H+共转运依赖于细胞膜内外的pH梯度,其pH梯度的维持正是质膜上的H+-ATPase的作用。竞争实验表明不同的阳离子不会降低Pi吸收速率,表明GmPT1和GmPT2对Pi吸收的高度特异性。含有GmPT1和GmPT2的菌株在毫摩尔浓度(mM)的Pi浓度下其吸收率与含空载的菌株相似。在32Pi吸收实验中,其吸收过程中的前5min吸收速率是线性的,利用Lineweaver-Burk图可以计算GmPT1和GmPT2的Km,得到GmPT1和GmPT2的Km分别为6.65mM和6.63mM。因此,GmPT1和GmPT2是低亲和PT并且依赖于质膜内外的质子梯度。5.播种7d后的大豆幼苗的根系、茎和叶片组织用于研究GmPT1和GmPT2的表达。在低磷胁迫的48h之内,幼苗的根系、茎和叶片中GmPT1和GmPT2的表达量都是升高的。48h高磷处理后,将幼苗进行低磷胁迫,3h后其幼苗的根系、茎和叶片中GmPT1和GmPT2的表达量也是上调的。用Hoagland营养液浇灌的幼苗移栽到高磷溶液后,其幼苗的GmPT1和GmPT2的表达量只有轻微的改变。48h低磷处理的幼苗移栽到高磷溶液3h后,其根系、茎和叶片中的GmPT1和GmPT2的表达量降低。然而,GmPT1和GmPT2基因的表达水平并没有受到磷浓度影响而显著地改变(其相对倍数变化是很小的)并且这种变化趋势是复杂的,这表明GmPT1和GmPT2是轻微诱导表达。6.酵母单杂交结果说明GmPHR1与GmSPX1的启动子存在着互作,这表明GmPHR1是通过顺式元件来和GmSPX1互作,因此GmPHR1可能是大豆Pi信号途径中的关键性调节子。更多还原

【Abstract】 Phosphorus is one of the most important macronutrients required for plant growth and metabolism, and is the key component of nucleic acids, phospholipids and ATP as well as several enzymes and coenzymes. Phosphorus (in the form of phos-phate) deficiency is the second most frequently limiting macronutrient for plant growth mainly because it exists in the soil in complex, insoluble, inorganic and or-ganic forms that cannot be acquired directly by the plant. It is well established that normal plant growth and productivity are dependent on the availability of inorganic phosphate (Pi). However, in many natural and agricultural ecosystems, plants often face conditions in which availability and mobility of phosphate are at lower extremi-ties. To cope with phosphate limitation, plants have evolved tightly controlled mecha-nisms to maintain phosphate homeostasis, which include the development of lateral roots and root hairs, as well as more dramatic root structures such as proteoid and dauciform roots, the secretion from roots of phosphatases and organic acids, and the induction of high-affinity and some low-affinity Pi transporters (phosphate transporter, PT). Many plants also establish symbiotic associations with mycorrhizal fungi that aid Pi acquisition. A variety of adaptive strategies have evolved in plants that alleviate or help them cope with Pi deficiency. The implementation of these strategies requires changes in the expression profiles of hundreds of genes. The extent and complexity of the network of regulatory genes necessary to sense Pi status and regulate the deploy-ment of these adaptive strategies is now being revealed. The network components identified so far include transcription factors, SPX sub-family proteins, non-coding RNAs and protein modifiers, including proteins involved in SUMOylation, phos-phorylation, dephosphorylation and protein translocation.In this study, we cloned a number of related genes, such as PTs, PHRs (phos-phate starvation response), SPXs (Syg1, Pho81, XPR1) and Gm4s; studyed on struc-ture of GmPTl and GmPT2with bioinformatics; analysed subcellular localization of GmPT1and GmPT2with green fluorescent protein fused to the two PTs; studied on functional and biochemical analysis of GmPT1and GmPT2in yeast mutant; analysed expression pattern of GmPT1and GmPT2with Real-time RT-PCR; studied on the interaction of GmPHR1and GmSPX1; At the same times, we used the principal com-ponents and membership function analysis to evaluate low phosphate tolerance of20soybean genotypes. The results are as follows:1. We used the principal components and membership function analysis to evaluate low phosphate tolerance of soybean genotypes. The tolerance (from strong-est to weakest) as follows:Gantai, Wuheqihuangdou,7650, Xiangdou No.4, Xianjin No.2, Su88M-21, Peking, Gaozuoxuan No.1,Kefeng No.1, Xinyixiaoheidou, Nannong1138-2,94-156,87-23, He84-5, Bogao, RN-9, Tongshanbopijia, Wan82-178, Xiangqiudou No.2, Dianjiangzaohuangdou.2. We identified two P; transporter genes in soybean located on chromosomes Gm10(41,391,168-41,393,008) and Gm20(42,980,124-42,981,928). These genes are designated GmPT1(accession number HQ392508) and GmPT2(accessionnum-ber HQ392509), respectively. GmPT1is1841-bp long and contains an open reading frame encoding a536amino acid polypeptide (molecular mass58730.46Da). GmPT2is1802-bp long and contains an open reading frame encoding a536amino acid polypeptide (molecular mass58627.29Da). Interestingly, the open reading frame in both genes spans base pairs23-1633. These genes are88.7%similar in nu-cleotide sequence and97.9%similar in amino acid sequence. The two polypeptides share the greater degree of similarity with the characterized PT from Arabidopsis, tomato, potato and barrel clover (Medicago truncatula). GmPT1and GmPT2have a very high degree of identity with fungal PT from the mycorrhizal fungus Glomus versiforme (GvPT) and the budding yeast Saccharomyces cerevisiae (PHO84). GmPT1shows76%and61%and Gm?T2shows76%and63%amino acid sequence identity with GvPT (accession number Q00908) and PHO84(accession number P25297), respectively. Hydropathy plots of the deduced polypeptides suggest that GmPT1and GmPT2consist of12membrane-spanning regions, a feature shared by PTs, irrespective of the level of affinity. Computational modeling of the encoded proteins predicted a conserved secondary structure containing12transmembrane (TM) domains with a large hydrophilic loop between TM6and TM7.3. The TBpred Prediction Server was used for searches that yielded unambi-guous results with positive scores for the integral membrane protein. To verify the subcellular locations of GmPT1and GmPT2, a green fluorescent protein (GFP)-tagged gene was fused to the3’end of the open reading frame of the GmPT1or GmPT2genes. A clear GFP signal was observed at the periphery of onion epi-dermal cells bombarded with the GmPTl/GFP or GmPT2/GFP construction, whe-reas the signal was seen throughout cells expressing free GFP. Localization of the GmPTl/GFP and GmPT2/GFP fusion proteins to the periphery of the cells indicated that the two proteins are targeted to the plasma membrane. This is consistent with the results of earlier biochemical studies and together these data suggest that the GmPT1and GmPT2proteins are located in the plasma membrane.4. We used uptake studies with inhibitors to confirm the pH dependence of Pi transport. Pi transport activity was assessed at pH values in the range4-7. Differ-ences were detected in the activity profiles but the uptake rate was maximal at pH4and increased as the pH was reduced from7to4in each case. To investigate this in-fluence of a proton motive force on Pi transport activity, the uncouplers2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), which destroy proton gradients across membranes, were applied. DNP at a concen-tration of100μM reduced the Pi uptake rate to79%(GmPT1) and82%(GmPT2) compared with100%uptake in the inhibitor-free control. The rate of uptake was re-duced to77%(GmPT1) and80%(GmPT2) by100μM CCCP and, to82%(GmPT1) and83%(GmPT2) by100μM Vanadate, an inhibitor of P-type H+-ATPases. The transporter rate was decreased significantly compared to that in the control. These results confirmed the hypothesis that Pi/H+cotransport via GmPT1and GmPT2de-pends on the pH gradient across the cell membrane that is maintained by the en-dogenous plasma membrane H+-ATPases. Moreover, competition studies showed that different anions did not reduce the Pi uptake rate, demonstrating the high degree of specificity of GmPT1and GmPT2for Pi. Strains carrying the GmPTl or GmPT2cDNA generally uptake Pi at rates similar to those of the vector controls at millimo-lar concentrations of Pi. In uptake experiments with radioactive Pi, the rate of trans-port was linear with time during the first5min of uptake under the conditions ap-plied. In three parallel experiments, the Lineweaver-Burk diagram, calculated using reciprocal uptake velocities at5min after addition of32Pi, indicated that Pi uptake facilitated by GmPT1and GmPT2followed Michaelis-Menten kinetics with an ap-parent Km valueof6.65mM and6.63mM, respectively. Thus, GmPTl and GmPT2are low-affinity Pi transporters that are dependent on the proton gradient across the plasma membrane.5. Root, stem and leaf tissues of7-day-old soybean seedlings were used to examine the expression of GmPT1and GmPT2. Expression of the two PT was en-hanced in both root and shoot during the first48h of Pi starvation. The expression of GmPTl and GmPT2in seedling tissues was increased during the3h after the Pi-sufficient treated seedlings were transferred to a P; deficient solution at48h com-pared to the expression measured in Pi-sufficient plants. The transcript levels of GmPT1and GmPT2were little changed in plants that were grown in half-strength nutrient solution for7days and then transferred to a Pi-sufficient solution. A de-crease in the transcript abundance of GmPT1and GmPT2in the leaf, stem and root of hydroponically grown soybean seedlings was apparent within3h of Pi deprivation. In conclusion, the expression level of GmPT1and GmPT2was not altered markedly and the change tendencies were complicated irrespective of how the seedlings were treated. Therefore, the soybean PTs GmPT1and GmPT2were slightly induced by P; deficiency.6. Interaction of GmPHRl with the promoter of GmSPXl was indicated by yeast one hybridization. The result indicated that GmPHR1can physically interact with GmSPXl through the cis-element, and this may be a key regulator in the Pi-signaling pathway in soybean.更多还原