苎麻谷氨酰胺合成酶基因的克隆和超量表达研究
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摘要
氮素是植物生长发育所必需的矿质营养元素，直接影响着植物的生长和发育，决定着作物产量的高低和品质的优劣。谷氨酰胺合成酶（GS）作为高等植物氮代谢途径中氮素初始同化中的关键酶，是一切无机氮素进入高等植物体内的“门户”，影响着植物氮素营养的吸收、同化及利用效率，对作物的生长发育，及产量、品质等农艺性状具有决定性作用。高等植物中谷氨酰胺合成酶分为胞液型（GS1）和质体型（GS2）两类：GS2主要同化NO-3还原而来及光呼吸过程所释放的氨；GS1主要同化从土壤吸收的和由NO-3还原而来的氨，及再同化从植物体内各个氮循环途径所释放的氨。苎麻（Boehmeria nivea L.）作为古老的纤维作物，近年来因其叶片中蛋白质及必须氨基酸含量较高、嫩茎叶营养价值与苜蓿相近、营养结构合理、年生物产量大等，被作为植物蛋白饲料原料而大量利用；同时，苎麻具有植株生长速度快、对氮素需求量大等特点。但是，国内外与苎麻氮代谢及生长特性相关的研究几乎为零，也没有进行苎麻氮代谢途径中功能基因的发掘，相关功能的研究和利用，从而阻碍了研究者对苎麻饲用及生长特性在本质上的了解，同时，也不利于苎麻中优良功能基因的发掘和利用。因此，本文以植物氮代谢途径中关键基因GS为研究对象，首次从苎麻中克隆获得GS基因家族中的4个基因，并利用生物信息学对苎麻BnGS基因序列和结构进行了分析，利用实时荧光定量PCR技术，分析了苎麻GS基因在不同组织和发育阶段的表达模式，同时，利用ClustalW和MEGA软件对GS基因进行序列比对和系统进化分析；另一方面，本文利用同源重组技术，构建了苎麻BnGS1-2基因的超量植物表达载体，并利用农杆菌侵染烟草叶片，成功获得转基因烟草植株，同时，研究了超量表达苎麻BnGS1-2基因后，转基因烟草植株对氮素的同化和利用效率，为了解苎麻GS基因的功能，及利用苎麻BnGS基因改良植物氮素利用效率和农艺性状的研究提供新的基因资源。本文主要研究结果如下：
（1）首次从苎麻栽培品种“中苎1号”中克隆获得苎麻GS基因家族中的4个基因，其中两个属于胞液型GS，命名为BnGS1-1和BnGS1-2；另外两个为质体型GS基因，命名为BnGS2-1和BnGS2-2，同时选取9株“中苎1号”自交后代，利用内切酶TaqⅠ酶切位点对两个质体型BnGS2基因进行TaqⅠ酶酶切鉴定，结果表明BnGS2-1和BnGS2-2为一对等位基因。
（2）利用生物信息学对苎麻GS基因序列和结构特征进行分析，结果表明BnGS1-1基因序列全长1205bp，含一个1071bp的开放阅读框（ORF），编码356个氨基酸残基多肽；BnGS1-2基因序列全长1222bp，起始密码子位于第123-125bp，终止密码子位于1102-1104bp，编码356个氨基酸残基多肽；两个BnGS2等位基因序列全长1340bp，含一个1293bp的ORF区，编码430个氨基酸残基多肽；通过序列比对发现BnGS2等位基因ORF区11个位点核苷酸存在差异，导致编码的多肽在195、382两个位点上的氨基酸存在替换现象；苎麻BnGS基因家族编码的多肽序列含有beta-Grasp和catalytic两个保守功能域，均属于Gln-synt结构域，同时，BnGS2基因编码的多肽序列含有一段信号肽；所克隆的苎麻BnGS基因家族编码的多肽序列在56、92、249和297位点上的氨基酸残基分别为天冬氨酸、半胱氨酸、组氨酸和谷氨酸，苎麻BnGS2等位基因在306和371两个位点上都为半胱氨酸残基。
（3）利用EMBOSS和MEGA软件对苎麻BnGS基因家族进行序列比对和系统进化分析，发现苎麻BnGS基因在蛋白质序列上的相似性为77.25-91.57%，在核苷酸序列上的相似性为71.15-79.37%，并且BnGS1两个基因之内要比BnGS1与BnGS2之间的相似性高。通过对苎麻和其它物种GS基因系统进化分析表明，BnGS1-2基因在进化上起源于双子叶植物，而BnGS1-1基因却被聚类在单子叶植物分支上，同时，苎麻BnGS1-2和BnGS2与苜蓿（Medicago sativa），大豆（Glycine max）和豌豆（Phaseolus vulgaris）等具有较近的亲缘关系。
（4）利用实时荧光定量PCR分析苎麻BnGS基因在不同部位和发育阶段的表达模式，结果表明BnGS1-1、BnGS1-2和BnGS2在苎麻叶片、茎、根、韧皮部及木质部中都有表达，但在不同的部位不同的BnGS基因的表达水平却呈现明显的差异。BnGS2基因主要的表达部位为叶片，同时，随着发育阶段的不同，相对表达量也呈现明显的不一样，表达量最高的时期为出苗期和纤维发育期；BnGS1-1基因在叶子、根和韧皮部中的相对表达量明显比茎及木质部高，但是在成熟阶段叶片中BnGS1-1的mRNA水平呈显著下降；韧皮部、木质部和茎中的BnGS1-2基因，在纤维发育时期被大量的诱导，表明苎麻BnGS1-2基因可能在纤维的发育过程中起着关键的作用。另一方面，BnGS1-1和BnGS1-2基因在苎麻整个生长过程中，在根部的表达量都呈现较高的水平。因此，苎麻不同的BnGS基因通过在特定部位表达量水平的调节，从而调控苎麻生长发育过程中对氮素营养的需要。
（5）本文利用同源重组技术将苎麻BnGS1-2基因转入pBI121植物表达载体特定位点，构建了能超量表达BnGS1-2基因的植物表达载体。在农杆菌（Agrobacterium tumefaciens）LBA4404的介导下，通过叶盘法将构建的超量表达载体转入烟草中。通过Kana筛选和基因组DNA PCR验证，获得转基因烟草植株。利用Q-PCR对转基因植株T1进行分析，结果显示BnGS1-2在转基因植株中检测到表达，转基因烟草中GS酶活性是野生型的1-2倍。超量表达BnGS1-2基因能显著增加转基因烟草植株的株高、鲜重和叶面积，因此，超量表达BnGS1-2基因能促进烟草植株的生长；另一方面，转基因植株体内水溶性蛋白与野生型烟草相比呈现极显著性升高，增长率达92.02%，总氮含量有所提高，但并没有达到显著性水平，同时，转基因植株体内游离NH+4含量显著下降，而游离NO-3含量与野生型相一致，因此，超量表达BnGS1-2基因，能够促进转基因烟草植株对氮素的吸收和利用，提高氮代谢效率，从而能够保证植株快速生长对氮素营养的需求。总之，本研究为苎麻GS基因功能的研究和应用提供一个理论和物质基础。
The mineral nutrients nitrogen is essential for higher plants growth, development and a majorlimiting factor in crops yield and quality. Glutamine synthetase (GS) plays fundamental roles in higherplants nitrogen primary assimilation and is regarded as the “hinge” over which inorganic nitrogen isconverted into organic form and transformed into plants. GS isoenzymes have essential effects to thenitrogen absorption, assimilation and use efficiency. Therefore, GS isoenzymes have critical functionsin growth and development, yield and quality of crops. According to their localization with the cell, GSisoforms include cytosolic (GS1) and plastid (GS2). The cytosolic isforms correlate with assimilation ofNH+4reduced from NO-3and re-assimilation of NH+4released from various metabolic pathways andGS2mainly exist in leaves with abundant chloroplast for incorporation of ammonium fromphotosynthesis and photorespiration. Ramie (Boehmeria nivea L.) is world-famous for its excellent fiberand commonly known as “China grass”. Simultaneously, ramie is regarded as ingredients for forageprotein due to equal nutritional value with alfalfa (Medicago sative), high protein and amino acidcontent, reasonable nutrition component and high biomass. In addition, ramie has special characteristicof growth having high rapid growth rate with4-6cm per day at vegetative stage. However, there haslittle or no physiology and molecular document been invested in understanding of ramie nitrogenassimilation and metabolism, and no functional genes in ramie nitrogen pathway have been excavatedand utilized. With the aim of gaining new insights in this area, especially at molecular levels, the focusof our study was first isolation and characterization of ramie GS gene families, and comparativelyanalyzed of GS gene families sequences, GS gene families expression pattern at different tissues anddevelopment stages and higher plants GS phylogeny. In addition, the over-expression plant vector oframie BnGS1-2gene was constructed according to homologous recombination technology andtransgenic tobacco was obtained by “leaf-disk” transformation method. Therefore, the investigation ofthe effects of BnGS1-2over-expression in improving the nitrogen absorption, assimilation and useefficiency provided theoretical basis at molecular level for ramie GS function and nitrogen assimilationpathway, and material basis for utilization of ramie GS genes. The main study results were indicated asfollows:
（1）Four genes of ramie GS gene families were first isolated from cultivator “Zhongzhu No.1”,two genes encoding cytosolic GS1and the other two encoding plastid GS2, named BnGS1-1, BnGS1-2,BnGS2-1, BnGS2-2, respectively. In addition, the two isolated BnGS2were allele genes, identified byTaq Ⅰ digestion of target genes in self-bred progenies, from ramie cultivator “Zhongzhu No.1”.
（2）Sequence and structural analysis showed that the cDNAof BnGS1-1gene with length of1205bp including a1071bp ORF region encoded polypeptide of356amino acids; the cDNA of BnGS1-2gene also encoded356amino acid polypeptide with length of1222bp including a1071bp ORF region.Two BnGS2allele genes encoded polypeptides of430amino acids with1340bp length including a 1293bp ORF region and a transit peptide. The diversity of nucleotide in11sites between BnGS2allelegenes resulting into amino acid residues substitution at site195and382. Four genes of ramie BnGSgene families contained beta-Grasp and catalytic functional domains which were belonged to Gln-syntdomain and conservative with other plants GS. The residues in site56,92,249,297of ramie BnGSgenes and in306,371of BnGS2allele genes were Asp, Cys, His, Glu, Cys and Cys respectively.
（3）Comparison of nucleotide and amino acid sequences showed that all ramie BnGS genefamilies shared very similar percentage in identity ranging from77.25-91.57%at protein sequence andat nucleotide levels with71.15-79.37%. The phylogenic analysis showed discrepant evolutionrelationship of ramie cytosolic GS genes with BnGS1-1grouped into monocots and BnGS1-2was asister to dicots. Simultaneously, the ramie BnGS1-2and BnGS2genes were similar with alfalfa(Medicago sativa), soybean (Glycine max) and bean (Phaseolus vulgaris) in phylogenic relationship.
（4）The expression patterns of ramie BnGS gene families at different tissues and developmentstages were investigated by fluorescence quantitative real-time PCR. The cytosolic and plastid BnGSgenes exhibited non-organ-specificity expression patterns but displayed very different transcriptionalconcentration. The main transcriptional tissue of BnGS2was leaves and gene expression levels variedalong with different development stages. BnGS1-1relative expression levels in leaves, roots and phloemwere higher than those in stems and xylem at all development stages. However, transcriptional levelswere remarkable reduced in leaves at mature stage. BnGS1-2relative expression levels weresignificantly triggered specifically in the phloem and xylem suggesting it has a primary role in ramiefiber development. On the other hand, BnGS1-1and BnGS1-2displayed high relative expression levelsin roots at all development stages. Therefore, the specificity in expression intensity rather than organ oframie different BnGS genes, may be a main factor to regulate nitrogen assimilation and metabolism inthe process of growth and development.
（5）The ramie BnGS1-2gene was ligated properly with plant expression vector pBI121toconstruct plant over-expression vector according to homologous recombination technology. Theover-expression vector was transferred into tobacco through Agrobacterium tumefaciens LBA4404using “leaf-disk” transformation method. Transgenic plants were obtained by Kana screening and DNAPCR determination. Q-PCR analysis showed BnGS1-2mRNA was detected in all transgenic tobacco T1plants. The GS enzyme activity in transgenic plant was detected at1-2times higher than wild type. Thetransgenic plants with over-expression BnGS1-2gene were significantly enhanced plant height, freshweight and leaf area suggesting BnGS1-2over-expression is able to promote the growth of transgenicplants. In addition, compared with wild type, the soluble protein of transgenic plants was remarkableenhanced up to92.02%, the total nitrogen content increased but did not reach significant level. TheBnGS1-2transgenic plants also exhibited an remarkable reduce of NH+4content, while the NO-3contentstill maintained the same levels with wild type. These results suggested that BnGS1-2over-expressionenable the transgenic plants to facilitate the nitrogen absorption, assimilation and use efficiency tosustain plants fast-growing demand for nitrogen nutrition. Therefore, the ramie BnGS1-2would be anexcellent gene resource for improvement of plants nitrogen utilization efficiency and crops agronomic traits.
（1）首次从苎麻栽培品种“中苎1号”中克隆获得苎麻GS基因家族中的4个基因，其中两个属于胞液型GS，命名为BnGS1-1和BnGS1-2；另外两个为质体型GS基因，命名为BnGS2-1和BnGS2-2，同时选取9株“中苎1号”自交后代，利用内切酶TaqⅠ酶切位点对两个质体型BnGS2基因进行TaqⅠ酶酶切鉴定，结果表明BnGS2-1和BnGS2-2为一对等位基因。
（2）利用生物信息学对苎麻GS基因序列和结构特征进行分析，结果表明BnGS1-1基因序列全长1205bp，含一个1071bp的开放阅读框（ORF），编码356个氨基酸残基多肽；BnGS1-2基因序列全长1222bp，起始密码子位于第123-125bp，终止密码子位于1102-1104bp，编码356个氨基酸残基多肽；两个BnGS2等位基因序列全长1340bp，含一个1293bp的ORF区，编码430个氨基酸残基多肽；通过序列比对发现BnGS2等位基因ORF区11个位点核苷酸存在差异，导致编码的多肽在195、382两个位点上的氨基酸存在替换现象；苎麻BnGS基因家族编码的多肽序列含有beta-Grasp和catalytic两个保守功能域，均属于Gln-synt结构域，同时，BnGS2基因编码的多肽序列含有一段信号肽；所克隆的苎麻BnGS基因家族编码的多肽序列在56、92、249和297位点上的氨基酸残基分别为天冬氨酸、半胱氨酸、组氨酸和谷氨酸，苎麻BnGS2等位基因在306和371两个位点上都为半胱氨酸残基。
（3）利用EMBOSS和MEGA软件对苎麻BnGS基因家族进行序列比对和系统进化分析，发现苎麻BnGS基因在蛋白质序列上的相似性为77.25-91.57%，在核苷酸序列上的相似性为71.15-79.37%，并且BnGS1两个基因之内要比BnGS1与BnGS2之间的相似性高。通过对苎麻和其它物种GS基因系统进化分析表明，BnGS1-2基因在进化上起源于双子叶植物，而BnGS1-1基因却被聚类在单子叶植物分支上，同时，苎麻BnGS1-2和BnGS2与苜蓿（Medicago sativa），大豆（Glycine max）和豌豆（Phaseolus vulgaris）等具有较近的亲缘关系。
（4）利用实时荧光定量PCR分析苎麻BnGS基因在不同部位和发育阶段的表达模式，结果表明BnGS1-1、BnGS1-2和BnGS2在苎麻叶片、茎、根、韧皮部及木质部中都有表达，但在不同的部位不同的BnGS基因的表达水平却呈现明显的差异。BnGS2基因主要的表达部位为叶片，同时，随着发育阶段的不同，相对表达量也呈现明显的不一样，表达量最高的时期为出苗期和纤维发育期；BnGS1-1基因在叶子、根和韧皮部中的相对表达量明显比茎及木质部高，但是在成熟阶段叶片中BnGS1-1的mRNA水平呈显著下降；韧皮部、木质部和茎中的BnGS1-2基因，在纤维发育时期被大量的诱导，表明苎麻BnGS1-2基因可能在纤维的发育过程中起着关键的作用。另一方面，BnGS1-1和BnGS1-2基因在苎麻整个生长过程中，在根部的表达量都呈现较高的水平。因此，苎麻不同的BnGS基因通过在特定部位表达量水平的调节，从而调控苎麻生长发育过程中对氮素营养的需要。
（5）本文利用同源重组技术将苎麻BnGS1-2基因转入pBI121植物表达载体特定位点，构建了能超量表达BnGS1-2基因的植物表达载体。在农杆菌（Agrobacterium tumefaciens）LBA4404的介导下，通过叶盘法将构建的超量表达载体转入烟草中。通过Kana筛选和基因组DNA PCR验证，获得转基因烟草植株。利用Q-PCR对转基因植株T1进行分析，结果显示BnGS1-2在转基因植株中检测到表达，转基因烟草中GS酶活性是野生型的1-2倍。超量表达BnGS1-2基因能显著增加转基因烟草植株的株高、鲜重和叶面积，因此，超量表达BnGS1-2基因能促进烟草植株的生长；另一方面，转基因植株体内水溶性蛋白与野生型烟草相比呈现极显著性升高，增长率达92.02%，总氮含量有所提高，但并没有达到显著性水平，同时，转基因植株体内游离NH+4含量显著下降，而游离NO-3含量与野生型相一致，因此，超量表达BnGS1-2基因，能够促进转基因烟草植株对氮素的吸收和利用，提高氮代谢效率，从而能够保证植株快速生长对氮素营养的需求。总之，本研究为苎麻GS基因功能的研究和应用提供一个理论和物质基础。
The mineral nutrients nitrogen is essential for higher plants growth, development and a majorlimiting factor in crops yield and quality. Glutamine synthetase (GS) plays fundamental roles in higherplants nitrogen primary assimilation and is regarded as the “hinge” over which inorganic nitrogen isconverted into organic form and transformed into plants. GS isoenzymes have essential effects to thenitrogen absorption, assimilation and use efficiency. Therefore, GS isoenzymes have critical functionsin growth and development, yield and quality of crops. According to their localization with the cell, GSisoforms include cytosolic (GS1) and plastid (GS2). The cytosolic isforms correlate with assimilation ofNH+4reduced from NO-3and re-assimilation of NH+4released from various metabolic pathways andGS2mainly exist in leaves with abundant chloroplast for incorporation of ammonium fromphotosynthesis and photorespiration. Ramie (Boehmeria nivea L.) is world-famous for its excellent fiberand commonly known as “China grass”. Simultaneously, ramie is regarded as ingredients for forageprotein due to equal nutritional value with alfalfa (Medicago sative), high protein and amino acidcontent, reasonable nutrition component and high biomass. In addition, ramie has special characteristicof growth having high rapid growth rate with4-6cm per day at vegetative stage. However, there haslittle or no physiology and molecular document been invested in understanding of ramie nitrogenassimilation and metabolism, and no functional genes in ramie nitrogen pathway have been excavatedand utilized. With the aim of gaining new insights in this area, especially at molecular levels, the focusof our study was first isolation and characterization of ramie GS gene families, and comparativelyanalyzed of GS gene families sequences, GS gene families expression pattern at different tissues anddevelopment stages and higher plants GS phylogeny. In addition, the over-expression plant vector oframie BnGS1-2gene was constructed according to homologous recombination technology andtransgenic tobacco was obtained by “leaf-disk” transformation method. Therefore, the investigation ofthe effects of BnGS1-2over-expression in improving the nitrogen absorption, assimilation and useefficiency provided theoretical basis at molecular level for ramie GS function and nitrogen assimilationpathway, and material basis for utilization of ramie GS genes. The main study results were indicated asfollows:
（1）Four genes of ramie GS gene families were first isolated from cultivator “Zhongzhu No.1”,two genes encoding cytosolic GS1and the other two encoding plastid GS2, named BnGS1-1, BnGS1-2,BnGS2-1, BnGS2-2, respectively. In addition, the two isolated BnGS2were allele genes, identified byTaq Ⅰ digestion of target genes in self-bred progenies, from ramie cultivator “Zhongzhu No.1”.
（2）Sequence and structural analysis showed that the cDNAof BnGS1-1gene with length of1205bp including a1071bp ORF region encoded polypeptide of356amino acids; the cDNA of BnGS1-2gene also encoded356amino acid polypeptide with length of1222bp including a1071bp ORF region.Two BnGS2allele genes encoded polypeptides of430amino acids with1340bp length including a 1293bp ORF region and a transit peptide. The diversity of nucleotide in11sites between BnGS2allelegenes resulting into amino acid residues substitution at site195and382. Four genes of ramie BnGSgene families contained beta-Grasp and catalytic functional domains which were belonged to Gln-syntdomain and conservative with other plants GS. The residues in site56,92,249,297of ramie BnGSgenes and in306,371of BnGS2allele genes were Asp, Cys, His, Glu, Cys and Cys respectively.
（3）Comparison of nucleotide and amino acid sequences showed that all ramie BnGS genefamilies shared very similar percentage in identity ranging from77.25-91.57%at protein sequence andat nucleotide levels with71.15-79.37%. The phylogenic analysis showed discrepant evolutionrelationship of ramie cytosolic GS genes with BnGS1-1grouped into monocots and BnGS1-2was asister to dicots. Simultaneously, the ramie BnGS1-2and BnGS2genes were similar with alfalfa(Medicago sativa), soybean (Glycine max) and bean (Phaseolus vulgaris) in phylogenic relationship.
（4）The expression patterns of ramie BnGS gene families at different tissues and developmentstages were investigated by fluorescence quantitative real-time PCR. The cytosolic and plastid BnGSgenes exhibited non-organ-specificity expression patterns but displayed very different transcriptionalconcentration. The main transcriptional tissue of BnGS2was leaves and gene expression levels variedalong with different development stages. BnGS1-1relative expression levels in leaves, roots and phloemwere higher than those in stems and xylem at all development stages. However, transcriptional levelswere remarkable reduced in leaves at mature stage. BnGS1-2relative expression levels weresignificantly triggered specifically in the phloem and xylem suggesting it has a primary role in ramiefiber development. On the other hand, BnGS1-1and BnGS1-2displayed high relative expression levelsin roots at all development stages. Therefore, the specificity in expression intensity rather than organ oframie different BnGS genes, may be a main factor to regulate nitrogen assimilation and metabolism inthe process of growth and development.
（5）The ramie BnGS1-2gene was ligated properly with plant expression vector pBI121toconstruct plant over-expression vector according to homologous recombination technology. Theover-expression vector was transferred into tobacco through Agrobacterium tumefaciens LBA4404using “leaf-disk” transformation method. Transgenic plants were obtained by Kana screening and DNAPCR determination. Q-PCR analysis showed BnGS1-2mRNA was detected in all transgenic tobacco T1plants. The GS enzyme activity in transgenic plant was detected at1-2times higher than wild type. Thetransgenic plants with over-expression BnGS1-2gene were significantly enhanced plant height, freshweight and leaf area suggesting BnGS1-2over-expression is able to promote the growth of transgenicplants. In addition, compared with wild type, the soluble protein of transgenic plants was remarkableenhanced up to92.02%, the total nitrogen content increased but did not reach significant level. TheBnGS1-2transgenic plants also exhibited an remarkable reduce of NH+4content, while the NO-3contentstill maintained the same levels with wild type. These results suggested that BnGS1-2over-expressionenable the transgenic plants to facilitate the nitrogen absorption, assimilation and use efficiency tosustain plants fast-growing demand for nitrogen nutrition. Therefore, the ramie BnGS1-2would be anexcellent gene resource for improvement of plants nitrogen utilization efficiency and crops agronomic traits.
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