【作者】 杨晓颖；

【导师】 郑绍建；

【作者基本信息】 浙江大学 ， 植物学， 2011， 博士

【摘要】 铝毒是限制酸性土壤上农作物生产的最主要的影响因子,植物的耐铝机制包括外部排斥和内部耐受,其中有机酸的分泌是植物重要的外部排斥机理,也是研究的热点。位于质膜上的有机酸转运子介导植物有机酸的分泌,是有机酸分泌的关键步骤。目前克隆到的有机酸转运子并不多,而且尽管有机酸分泌具有明显的A1诱导特征,但迄今还没有发现完全被铝诱导表达的有机酸转运子。我们实验室前期研究发现,A1胁迫可诱导饭豆根尖柠檬酸的分泌,且其分泌起始与施加A1胁迫之间有一个明显的滞后期。本研究通过同源克隆手段从饭豆中克隆得到了该柠檬酸转运子,并对其表达和功能进行了研究。另一方面,尽管有机酸的分泌是植物重要的耐铝机理,但不能解释水稻的铝耐性,本研究还以水稻为材料研究了果胶甲酯酶基因家族与铝诱导的根伸长受抑制间的关系。主要结果如下：1.从头合成的柠檬酸转运子VuMATE在饭豆铝耐性中的作用饭豆(Vigna umbellata)根系在铝胁迫下可专一性的分泌柠檬酸,并且分泌被延迟了数小时。本研究采用同源克隆和RACE技术从饭豆根尖中分离了一个从头合成(新合成)的柠檬酸转运子基因,由于其属于MATE基因家族的成员,将其命名为VuMATE。 VuMATE编码一个565个氨基酸的蛋白质。VuMATE与己知的柠檬酸转运子具有高度的序列相似性,都包含12个跨膜结构域,在跨膜域2和3之间都有一个位于细胞质中的环状结构,此结构也包含一段保守的氨基酸序列,推测其在柠檬酸转运方面具有重要作用。多序列比对显示,VuMATE与白羽扇豆的LaMATE亲缘关系最近。RT-PCR分析表明VuMATE在铝处理前没有表达,是第一个完全被铝诱导表达的有机酸转运子基因。VuMATE的表达部位仅限定在0-1cm根尖(铝毒的靶位),在1-2cm的根成熟区和叶片中都不表达。VuMATE的表达量随着A1处理浓度和处理时间的增加而增加,这与饭豆铝胁迫下根系柠檬酸的分泌模式相一致。VuMATE的表达不受二价金属Cd和Cu的诱导,三价金属La可以诱导VuMATE的表达,但同时加入A1会使表达量进一步提高。亚细胞定位显示VuMATE是一个膜蛋白。将VuMATE异源表达于爪蟾卵母细胞中的功能分析表明,VuMATE可以介导显著的跨越质膜的内向电流,内向电流的强度依赖于细胞外的H+离子(也可能是Na+)。注射14C标记的柠檬酸发现,VuMATE可以直接介导14C柠檬酸的转运,其转运能力也依赖于细胞外H+。通过转基因的方法在番茄中过表达VuMATE赋予了番茄分泌柠檬酸的能力,提高了番茄对铝的耐受性。所有这些结果说明VuMATE是饭豆中负责柠檬酸分泌的柠檬酸转运子。通过比较饭豆柠檬酸转运子与其它已知柠檬酸转运子的异同,发现了饭豆柠檬酸转运子具有不同于其它柠檬酸转运子的特点。2.水稻果胶甲酯酶基因与铝诱导的根伸长抑制相关水稻是小粒谷类中最耐铝的物种,但有机酸的分泌不能解释其强耐铝性,有研究表明铝主要和细胞壁果胶的负电荷结合,而果胶的负电荷的产生与果胶甲酯酶密切相关。为此,我们通过对水稻果胶甲酯酶基因家族的表达分析探讨了其与铝诱导根伸长抑制的关系。从DFCI数据库中下载了水稻果胶甲酯酶基因家族的35个成员,其cDNA序列的GC含量很高,每个成员具有不同的基因结构,所编码的蛋白质分为两组(一组只有PME结构域,一组在PME结构域前还有PMEI结构域),系统发生关系显示水稻果胶甲酯酶可以分为5类。当用25μM Al处理铝敏感水稻品种浙辐802("Zhefu802")3h,根伸长被抑制了40%,根尖细胞壁的PME活性提升了20%。这时,35个PME基因的表达变化分为4组：A组不表达,B组上调表达,C组下调表达,D组表达量不变。由于B组上调表达的8个PME基因与其酶活变化一致,我们初步认为这8个基因与铝诱导的根伸长抑制相关。我们进一步在铝耐性水稻品种日本晴("Nipponbare")中验证了筛选出的8个基因与铝胁迫的关系。结果表明：25μM Al处理3h不影响日本晴根伸长,此时,这8个基因的表达也不受影响；但当将Al处理浓度提高到50μM Al时,日本晴的根伸长受抑制也达到40%,此时,除了2个不表达的基因外,其它6个基因的表达都明显上调。由此,进一步证明了这8个基因与铝诱导的根伸长抑制相关。8个基因对其它金属胁迫的响应不同于铝胁迫,暗示着不同的PME基因对不同的金属胁迫做出响应。综上所述,我们从水稻果胶甲酯酶基因家族中筛选出了8个与铝诱导的根伸长抑制相关的基因,为从分子水平上研究细胞壁在抗铝胁迫中的作用提供了切入点。更多还原

【Abstract】 Aluminum (Al) toxicity is one of the most important deleterious factors limiting crop production on acid soils. Al resistance mechanisms in plant have been classified into external exclusion and internal tolerance. Among them, secretion of organic acid anions is an important external exclusion mechanism. The transporter on the plasma membrane mediates the secretion of organic acid anion, which is the crucial step of the secretion. However, only a few organic acid anion transporters have been cloned by now, and transporter induced completely by Al stress hasn’t been isolated although the secretion of organic acid anion has the obvious Al-induced characteristics. Our previous study showed that citrate secretion could be induced with a lag phase after the initiation of Al stress in rice bean. Therefore, the aim of this study was to clone the citrate transporter responsible for citrate efflux from rice bean. The function of the transporter was also investigated. On the other hand, the secretion of organic acid anion cannot explain the Al resistance mechanism in some plants. Thus, another focus of our research was to investigate the relationship between cell wall and Al resistance in rice as it had been demonstrated that Al-induced organic acid anions secretion was not involved in high Al resistance in rice. We tried to associate some specific pectin methylesterase genes with Al-induced root elongation inhibition in rice. The results were summarized as following:1. A de novo synthesis citrate transporter VuMATE confers aluminum resistance in rice bean(Vigna umbellata)Al specifically induces citrate efflux in rice bean, and the efflux is delayed for several hours. Here we conducted homologous cloning and RACE technology to isolate a gene encoding a de novo synthesis (newly synthesized) citrate transporter from the root tip of rice bean, which belongs to MATE gene family and was named as VuMATE(Vigna umbellata multidrug and toxic compound extrusion). The deduced protein of VuMATE comprised565amino acids.VuMATE showed high protein sequence homolog to the known citrate transporters. They all are twelve-span transmembrane proteins and share a common highly conserved amino acid sequences in intracellular loop between the2nd and3rd transmembrane domain. The intracellular loop is cytoplasmic and unique to the known citrate transporters of MATE family, so it is possible that the conserved sequences in the loop play an important role in citrate transport. Phylogenetic relationship analysis showed that VuMATE clustered most nearly with LaMATE from white lupin.In the absence of Al, the VuMATE mRNA transcription was not detected neither in root tip nor leaf tissue. Al treatment induced the expression only in the root segment0-1cm from the root tip, while still no expression could be detected in the1-2cm region and nor in leaf tissue. Al-induction of VuMATE expression was both Al-concentration and time dependent, increasing at higher Al levels and durations of Al exposure, which was consistent with citrate secretion pattern. To determine the Al-specificity of VuMATE induction of expression, the effect of the other metals was tested. While cadmium (Cd) and copper (Cu) alone didn’t induce VuMATE expression, lanthanum (La) induced VuMATE expression in an additive manner, in that VuMATE expression in response to La+Al was higher than in response to La alone.The transient expression assays with a VuMATE::GFP translational fusion in onion epidermal cell showed that VuMATE localized at plasma membrane.Electrophysiological analysis of Xenopus oocytes expressing VuMATE indicated this transporter could mediate significant anion efflux across the plasma membrane. VuMATE-mediated inward currents were pH dependent (and possibily Na+dependent). After injecting14C-labeled citrate, VuMATE-expressing cells showed enhanced14C efflux, indicating that VuMATE could mediate citrate efflux directly. Moreover,14C efflux from cells expressing VuMATE was also pH dependent.Overexpression of VuMATE in transgenic tomato conferred citrate efflux in root and consequently Al resistance increase.All these indicated that VuMATE was the citrate transporter responsible for citrate efflux in the root apex of rice bean. By comparing the citrate transporter in rice bean with the other known citrate transporters, we summarized some specific characteristics possessed by the citrate transporter in rice bean. 2. Association of specific pectin methylesterase genes with Al-induced root elongation inhibition in riceRice (Oryza sativa L.) is the most Al resistance species among small-grain cereal crops. The secretion of organic acid anion cannot explain its Al resistance. It has been reported that the negative charges of cell wall pectin molecules determined by pectin methylesterase (PME, EC3.1.1.11) contribute to Al binding. Therefore, this study focused on the relationship between PME gene family with Al-induced root elongation inhibition in rice.Thirty-five putative Oryza sativa L. PME gene sequences were retrieved from DFCI (http://compbio.dfci.harvard.edu/tgi/plant.html. The GC%of PME genes in rice is very high. Each member has different gene structure. Their deduced proteins were classified as group1(only having a PME domain) or group2[having a PMEI (PME Inhibitor) domain preceding the PME domain]. The phylogenetic tree analysis showed that there were five distinct clades (Clade I to V) in rice.After treatment Al-sensitive rice sp. indica’Zhefu802’with25μM Al for3h, root elongation was inhibited by40%, and CW PME activity was increased by20%. Meanwhile, the expression pattern of PME could be divided into4classes. Nine of PME genes had no expression signals (Class A), eight genes were up-regulated by Al (Class B), two genes were down-regulated (Class C), and the remainder exhibited constitutive expression pattern which was not affected by Al treatment (Class D). As the result of the consistency between8up-regulated genes with PME activity increase, it could be speculated that the eight up-regulated PMEs might be associated with Al-induced root elongation inhibition in rice.We further investigated the eight genes’expression pattern in an Al-resistant rice cultivar sp. japonica’Nipponbare’. While root elongation of rice’Nipponbare’was not significantly affected by25μM Al treatment for3h, the expression of these eight genes was not affected. However, when Al concentration was increased to50μM, by which the root elongation was also inhibited by40%, the expression of the eight PME genes was up-regulated except two no signal genes. These results suggested that the eight PME genes were likely related to the Al-induced inhibition of root elongation both in Al-sensitive and Al-resistant rice cultivars.The eight PME genes behaved differently under CdCl2and LaCl3treatment, implying the specificity of different PME genes in response to different metal toxicities. Altogether, we chose the eight members from PME gene family to be associated with Al-induced inhibition of root elongation in rice. Our results provided a breakthrough point to study the effect of Al stress on cell wall.更多还原