【作者】 邓扬悟；

【导师】 黄丹枫；

【作者基本信息】 上海交通大学 ， 蔬菜学， 2010， 博士

【摘要】 谷氨酰胺合成酶(GS; EC 6.3.1.2)是植物N素同化途径中最为关键的催化酶之一,被称为是植物中无机态N转化为有机态N的“门户”,对植物N素吸收、同化和利用效率(Nitrogen use efficiency)有着极为重要的影响。高等植物中的GS同工酶主要分为两类:胞质型GS1主要同化从土壤吸收的初级氨及再同化从植物体内各个N循环途径所释放的氨;质体型GS2同化由NO3--N还原而来及光呼吸过程所释放的氨。N素供应对甜瓜的生长发育、果实的产量和品质形成有非常重要的影响,目前甜瓜N素代谢研究还停留在N营养生理与果实品质及产量层面,在分子机理水平的研究报道很少,尤其是对与N素同化和利用效率紧密相关的GS酶基因的研究还是空白。因此,本文以甜瓜作为对象,在从甜瓜中克隆出首个胞质型GS基因M-GS1的基础上,对M-GS1及课题组克隆到的首个甜瓜质体型GS基因M-GS2的基因组拷贝数、表达产物的亚细胞定位及生化特性、在甜瓜中的表达调控特征等进行了研究和对比分析,从基因、蛋白质和细胞水平对甜瓜GS基因进行了系统的功能验证和鉴定,开展了甜瓜N营养代谢的分子生理研究;进而在植株水平研究M-GS1在转基因超量表达后提高植株N素同化效率的潜能,为甜瓜GS基因的应用、利用GS基因改良植物N素利用效率的研究提供新的材料和依据。主要研究结果如下。1、甜瓜胞质型谷氨酰胺合成酶基因的克隆及生物信息学分析本研究采用RACE-PCR方法从甜瓜(Cucumis melo L. var. reticulatus Naud.)品种“春丽”中克隆出首个GS1基因M-GS1(GenBank登录号: DQ851867)的全长cDNA序列,并利用生物信息学手段分析其主要结构和功能特征。该基因全长1494 bp,含一个1068 bp的开放阅读框(ORF),推测编码一个由356个氨基酸残基组成的多肽。氨基酸序列比对结果显示,M-GS1与源自其它植物的GS1有较高的同源性;氨基酸序列分析显示该蛋白具有植物GS同工酶的典型结构特征:内含一个GS beta-Grasp域和一个GS catalytic域,以及分别存在于这两个域中的两个活性中心(motif)——一个GS指纹区和一个GS ATP结合区。对M-GS1三级立体结构的预测分析显示,它与通过X光衍射鉴定的晶体GS结构一致,其立体结构的正确折叠需要有Mn2+、AMP及Citric acid等3个配体的存在。系统进化树分析表明,M-GS1与北美假大麻(Datisca glomerata)GS1在分子进化关系上最相近,提示二者可能是同源进化的结果。2、甜瓜谷氨酰胺合成酶基因的基因组拷贝数对M-GS1及课题组克隆到的首个甜瓜质体型GS基因M-GS2(GenBank登录号: AY773090)在甜瓜基因组中的拷贝数进行了对比分析。Southern blot结果显示,在甜瓜基因组中,M-GS1可能被一个由2到3个基因成员组成的基因家族所编码,而M-GS2则由单个基因编码。3、甜瓜谷氨酰胺合成酶的亚细胞定位通过将甜瓜M-GS1及M-GS2基因分别与报告基因——黄色荧光蛋白YFP基因融合,构建重组植物表达载体(pA7-GS1::YFP和pA7-GS2::YFP),应用基因枪法转入洋葱内表皮细胞中进行瞬时表达,然后在激光扫描共聚焦显微镜下,对表达产物在洋葱内表皮细胞内的分布情况进行了观察和分析。结果显示,融合蛋白M-GS1::YFP定位于洋葱内表皮细胞的细胞质中,而融合蛋白M-GS2::YFP则定位于洋葱内表皮细胞的质体中。这些结果在细胞水平证实了甜瓜M-GS1和M-GS2蛋白确实具有其自身所属同工酶类别(GS1或GS2)通常所应具有的亚细胞定位特性。4、甜瓜谷氨酰胺合成酶基因的原核表达及产物GS酶活性相关分析为了对甜瓜M-GS1及M-GS2各自所编码的GS酶的生化功能和属性有准确的了解,本实验分别对M-GS1编码区、M-GS2编码区及去除跨肽cTP序列的M-GS2编码区(M-GS2-cTP)成功地进行了原核表达,用Ni–NTA亲和色谱法纯化得到了各对应的重组蛋白。SDS-PAGE电泳结果显示,重组M-GS1、M-GS2和M-GS2-cTP蛋白的分子量分别为41 kDa、50 kDa和44 kDa。分析各重组蛋白质的谷氨酰胺合成酶催化特性显示,只有重组M-GS1蛋白质和重组M-GS2-cTP蛋白质具有GS酶催化活性,而未切除cTP序列的重组M-GS2蛋白质无GS催化活性;M-GS1比M-GS2-cTP具有更高的NH4+亲和性和GS酶合成酶活性,但其热稳定性相对更低;两种甜瓜GS酶的活性明显受Mg2+浓度的调节。这些结果进一步在蛋白质水平证实M-GS1和M-GS2为谷氨酰胺合成酶基因,而它们的表达产物具有各自不同的生化特性。5、不同形态N营养施养处理对甜瓜中谷氨酰胺合成酶基因表达的调控用实时荧光定量PCR方法,分析不同形态N素施养处理对甜瓜M-GS1及M-GS2表达的潜在调控作用。结果显示,不同形态的N素施养处理对甜瓜中M-GS1及M-GS2的表达呈现不同的调节模式。NH4+-N和NO3--N中、低浓度(3.75 mM或0.75 mM)施养处理仅对甜瓜叶片中M-GS2的表达有显著的调节作用;足量的N素营养浓度水平(7.5 mM)施养处理则对M-GS2表达量无明显调节作用。然而,谷氨酸施养处理,不仅调节M-GS2在叶片中的表达(开始降低而后升高到一个比对照高的水平),还调节其在根中的表达;对甜瓜根中M-GS1表达产生抑制作用,对其在叶片和茎中的表达则无明显影响。NH4+-N施养处理能刺激M-GS1基因在甜瓜苗根、茎、叶中的表达,而NO3--N则仅刺激其在甜瓜根和叶中的表达。以上结果表明,正是由于不同的GS同工酶基因有不同的表达调节特性,具有差异化的生理功能,才能保证植物在应对现实生长环境中不断变化的N营养供应状况时其N素同化作用得以正常进行。6、甜瓜M-GS1转基因超量表达对拟南芥N相关生理生化的影响将甜瓜M-GS1基因正确构建于植物表达载体pEZT-NL中,获得含目的基因的植物表达载体EHA105-35S::M-GS1::EGFP。在根癌农杆菌(Agrobacterium tumefaciens)EHA105的介导下,通过浸花法(Floral-dip)再将构建的植物表达载体转入拟南芥。通过除草剂筛选及基因组DNA PCR验证,获得了十个独立的转M-GS1基因株系。提取独立转基因株系总RNA进行RT-PCR分析,结果显示,M-GS1在各独立转基因株系中均检测到了表达。正常(7 mM)及低N素(1mM)水平培养实验发现,转M-GS1基因对植株生长生化指标的影响只在低N素水平培养时显著表现。生化及生长分析显示,在低N素供应时,各M-GS1转基因株系莲座叶(rosette)的总GS酶活性、总可溶性蛋白含量和生物量均显著地高于野生型拟南芥植株。这些结果表明,M-GS1基因的超量表达具有在低N素供应环境下维持转基因植株N素同化利用效率的潜能。更多还原

【Abstract】 Glutamine synthetase (GS; EC 6.3.1.2) plays fundamental roles in plant N assimilation and is regarded as the‘hinge’over which inorganic N is converted into its organic form. GS isoenzymes have demonstrated central effects to the enhancement of plant N use efficiency. According to their localization within the cell, GS isoenzymes in higher plants are classified into 2 groups, including GS1, the cytosolic forms that localize in cytosol responsible for assimilation of the NH4+ directly uptake from soil by roots and for re-assimilation of the NH4+ released from various metabolic pathways, and GS2, the plastid forms that function in cell plastids for incorporation of the NH4+ reduced from NO3- and that released from photorespiration process into glutamine.Not only the growth and development but also the ultimate yield and fruit quality of melons were reported significantly regulated by supplementation of N nutrition. However, our knowledge of N assimilation processes in melons is still very limited, and previous studies were basically contrained at the levels of plant physiology and yield and fruit quality, and reports on isolation and characterization of GS genes, the key player of plant N metabolism, in melons are not documented. With the aim of gaining new insights in this area, especially at molecular levels, in this study, we cloned and characterized the first cytosolic GS gene from melon, M-GS1 (GenBank accession No.: DQ851867), and comparatively analyzed at molecular levels the genomic copy number, gene products localizations within the cell, the GS activities of the corresponding recombinant proteins expressed and purified from E. coli, and the expression patterns of M-GS1 and M-GS2 (GenBank accession No.: AY773090), a GS2 gene previously cloned in melon by our group regulated, by various forms of N nutrients, and in addition, investigated at plant level the effects of M-GS1 over-expression in improving the nitrogen use efficiency of the transgenic Arabidopsis lines. The main results were indicated as follows: 1. The cloning and bioinformatics’analyses of M-GS1RACE-PCR techniques were employed to successfully clone the first GS1 gene from melon(Cucumis melo L. var. reticulatus Naud.) M-GS1 (GenBank accession No.: DQ851867). The full-length cDNA of M-GS1 contains 1494 nucleotides with an open reading frame (ORF) of 1068 nucleotides. The deduced 356 amino acid sequence showed high similarity with previously reported GS1 isoenzymes from various plant species. Sequence analysis revealed that the predicted protein contains a GS beta-Grasp domain, a GS catalytic domain, and the main conserved motifs characteristic of a plant GS1. The phylogenetic analysis displayed that M-GS1 is related most closely to the GS1 from Datisca glomerata evolutionarily. The predicted 3-D structure for M-GS1 indicated that it conforms structurally to the X-ray crystallogram of a typical GS subunit and requires the involvement of bi-valent metal ions including Mn2+ or Mg+, AMP and citric acid to fold into its active conformations.2. The genomic copy number of M-GS1 and M-GS2 in melonAs shown by Southern blot analysis, similarly but relatively simpler to the results found in other plants, melon genome contains 2-3 copies of M-GS1, the cytosol GS genes, but one copy of M-GS2, the chloroplastic GS gene.3. The sub-cellular localization analyses of M-GS1 and M-GS2In this experiment, we constructed two recombinant plant expression plasmids, in which both M-GS1 or M-GS2 was fused with a fluorescence protein YFP, the reporter gene, and the fusion was driven by a CamV 35S promoter. The recombinant plant expression plasmids were then transferred into the onion epidermal cells via the bombardment using a gene gun, and expressed transiently. Fluorescence emissing from the expressed products localizing in different cell organelles was visualized and analyzed using laser-scanning confocal microscope techniques. The results showed, that while M-GS1::YFP fusion proteins were localized in the cytosol, M-GS2::YFP fusion proteins were localized in plastids, confirming that both M-GS1 and M-GS2 indeed bears the sub-cellular localization characteristics of the corresponding GS group that they were assigned to as a member, respectively.4. M-GS1 and M-GS2 expression in E. coli and GS activity related analyses of the purified recombinant proteinsTo characterize the biochemical properties of M-GS1 and M-GS2, we constructed 3 recombinant expression plasmids to express the coding sequences of either M-GS1, M-GS2, or M-GS2 minus cTP(M-GS2-cTP) sequences in E. coli., respectively. As shown by SDS-PAGE and biochemical analyses, all the 3 recombinant proteins, purified using Ni-NTA affinity chromatography, were properly expressed, but only M-GS1 and M-GS2-cTP were found having GS activities. Activity analysis showed that M-GS1 has evidently higher affinity for NH4+ and higher synthetase and transferase activity than M-GS2-cTP but is less resistant to heat, and Mg2+ was found playing a significant role in enhancing the GS activities of both recombinant proteins. These results provided further evidences that the putative M-GS1 and M-GS2 genes cloned in melon were indeed GS genes that express GS isoenzymes of differential biochemical properties.5. The regulation of M-GS1 and M-GS2 expression by various forms of N nutrients in fertilizationReal-time quantitative PCR was performed to investigate the potential differential regulations of M-GS2 and M-GS1 expression by various forms of N nutrients in fertilization. Results showed that distinct forms of nitrogen (N) found in fertilizers transcriptionally regulated M-GS2 differently. Ammonium and nitrate feeding only significantly regulated M-GS2 transcripts in leaf; starving (0.75 mM) or moderate (3.75 mM) N levels dramatically increased M-GS2 transcripts for 1 day, decreasing to a constant low level after 2-3 days, while sufficient N level (7.5 mM) had a minor effect throughout 3 days compared to controls. Glutamate feeding, however, not only significantly regulated M-GS2 transcripts in leaf (decreased initially then increased to higher levels than controls), but also in root, where it was up-regulated continuously. M-GS1 was expressed in all plant tissues without evident tissue specificity, but with differential patterns when the melon plants were fed in hydroponic culture with different forms of N nutrient at differential N concentrations: ammonium dramatically enhanced the levels of M-GS1 transcripts in all tested tissues, while nitrate stimulated M-GS1 transcription only in the roots and leaves, but not in the stems; Glu (glutamate), however, depressed M-GS1 transcripts in the roots, but resulted in no significant change to the levels of M-GS1 transcripts in the stems and leaves. These results suggest that to enable sustained N assimilation under different conditions of N availability melon responded by differentially regulating the expression of GS genes into GS isoenzymes of differential physiological functions.6. The N related effects of M-GS1 over-expression on transgenic Arabidopsis linesThe coding sequence of M-GS1 cDNA was ligated properly with plant expression vector pEZT-NL to construct a plant expression vector driven by CAM 35S promoters that expresses a fusion protein M-GS1::EGFP. This expression vector was transferred into Arabidopsis via Agrobacterium tumefaciens EHA105 using the‘Floral-dip’method. Ten independent transgenic plant lines were obtained through PPT screening and DNA PCR verification. RT-PCR analysis showed M-GS1 expression were detected in all independent transgenic lines. As shown in N-level-dependant growth experiments, the effects of M-GS1 over-expression were evident only when N was supplied at low levels. As indicated by biochemical and growth indicators, all transgenic lines displayed evidently higher rosette total GS activity, total soluble protein content and biomass than wild-type plants under low N supply. These results suggested that M-GS1 over-expression enabled the transgenic plants to sustain relatively higher levels of N assimilation and growth under N limited conditions than wild-type plants, and has the potentials in improving plant N use efficiency.更多还原