【作者】 卢永恩；

【导师】 张启发； 练兴明；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 氮是植物必需的大量元素,对植物的生长发育至关重要,也是决定作物产量的重要因子。近年来,氮肥的大量施入虽然增加了作物产量,但对生态环境也造成了巨大的破坏。通过现代生物技术提高作物的氮素利用率(NUE),是解决粮食增产和环境保护这一矛盾的根本出路。水稻是我国最重要的粮食作物之一,其氮肥需求量巨大,提高水稻的NUE意义重大。充分了解氮素吸收和同化关键基因的生物学功能,对于深入解析碳氮代谢的分子机制,进而通过精细调控提高作物NUE至关重要。谷氨酰胺合成酶-谷氨酸合酶(GS-GOGAT)循环是氮素同化过程中无机氮转变为有机氮的起点,也是碳氮结合的交叉点,在提高作物NUE工程中的重要性不言而喻。目前,人们对GOGAT基因在氮素吸收和碳氮代谢中扮演的角色仍未形成一个清晰的认识,GS-GOGAT循环所需碳骨架(α-酮戊二酸,20G)的供应机制也未得到解析。本研究即以获得的NADH-GOGAT及胞质ICDH (NADP-依赖型异柠檬酸脱氢酶)受到抑制的两类转基因水稻为材料,从主要代谢产物变化、相关酶活水平以及相关基因表达等方面进行了分析,对NADH-GOGAT的生物学功能以及胞质ICDH在提供20G过程中的角色定位问题进行了深入的探讨。同时,本研究还对水稻氨基酸转运蛋白(OsAAT)基因家族进行了生物信息学分析和转录表达分析,获得了其中11个成员的T-DNA插入突变体,并对其碳氮含量进行了初步分析。主要结果如下：1.在粳稻中组成型表达OsNADH-GOGAT基因导致OsNADH-GOGAT1和OsNADH-GOGAT2的共抑制,OsFd-GOGATl也下调表达。共抑制家系在正常营养供应的田间表现为氮缺乏症状,如分蘖减少、株高变矮、产量和千粒重下降。代谢物检测显示,NADH-GOGAT共抑制家系叶片的可溶性糖、总氮以及多数含氮化合物含量下降,20G、异柠檬酸和游离氨含量增加,部分碳氮代谢相关酶活也受到影响。这表明,NADH-GOGAT在水稻碳氮代谢的多个层面发挥着重要作用,是水稻氮素高效利用的关键基因；2.在粳稻中组成型表达OsICDH2导致共抑制,使OsICDHl和内源OsICDH2表达量降低,叶片和根部ICDH酶活分别较野生型降低43.5%-76.6%和19.4%-56.4%。正常营养条件下,ICDH共抑制家系表现为生长量减少,田间产量降低,表明胞质ICDH是保持水稻高产稳产的重要因子；3.当置于高温下3-6天,ICDH共抑制家系幼苗逐渐失绿并最终死亡,而野生型表现正常。高温下,与野生型比较,共抑制家系叶片中的叶绿素、多种有机酸、可溶性糖、硝酸盐含量降低,游离氨、总游离氨基酸、天冬酰胺和葡萄糖-6-磷酸含量增加,谷氨酸脱氢酶、天冬氨酸转氨酶、葡萄糖-6-磷酸脱氢酶、果糖激酶和葡萄糖激酶活性上升,GS活性下降,OsGDHl和OsGDH2上调表达,OsGS2和OsFd-GOGAT1下调表达。我们推测,水稻IDH (NAD-依赖型异柠檬酸脱氢酶)和胞质ICDH均可以为氮素的同化提供20G,IDH是20G的主要来源,胞质ICDH仅在特殊条件下,如高温胁迫期间,20G供应不足时,提供必要补充。谷氨酸脱氢酶、天冬氨酸转氨酶以及20G转运蛋白均参与了20G的补充过程；4.共鉴定出65个OsAAT基因,其中49个受到全长cDNA支持,部分OsAAT基因具有器官特异或氮饥饿诱导的表达模式。获得了11个OsAAT成员的T-DNA插入突变体,与野生型比较,部分突变体的碳氮含量发生了显著改变,表明这些基因与水稻的碳氮分配关系密切。更多还原

【Abstract】 Nitrogen is an essential macroelement for plant growth and development, and is one of the most important limiting factors in crop production. In recent decades, luxury application of nitrogen fertilizer greatly enhanced crop production. However, it also caused severe environmental contamination. Contradictorily, demands for food are still increasing because of the increasing population in the world. To solve this problem, improving crop nitrogen use efficiency (NUE) through biotechnology is a smart choice. Rice is the staple food of China and the demand of nitrogen fertilizer in its production is huge, it is of great significance to improve the NUE of this crop.It is valuable to thoroughly identify the biological functions of key genes of nitrogen absorption and assimilation for understanding the molecular mechanism of carbon and nitrogen metabolism and improving crop NUE. The glutamine synthetase-glutamate synthase (GS-GOGAT) pathway is located at the cross-point of carbon and nitrogen metabolism, suggesting its important role in NUE improving engeering, through which inorganic nitrogen can be transformed into organic nitrogen. Researches focused on GOGAT genes are less, which lead to an indistinct understanding of its fuction in nitrogen assimilation. Another problem in GS-GOGAT pathway is that the origin of carbon skeleton (2OG, a-ketoglutarate) provided for this pathway in nitrogen assimilation is still unclear. In this research, biological functions of NADH-GOGAT in carbaon and nitrogen metabolism and the roles of cytosolic ICDH (NADP-dependent isocitrate dehydrogenase) in providing2OG for ammonium assimilation were systematically investigated, through testing the carbon and nitrogen metabolites contents, related enzyme activities and selected genes transcription levels, using NADH-GOGAT or cytosolic ICDH cosuppressed transgenic rice plants. In addition, molecular characterization, expression and functional analysis of rice amino acid transporter (OsAAT) gene family were also conducted in this research. The main results are as follows:1. Constitutive expression of OsNADH-GOGAT in japonica rice caused cosuppression of both OsNADH-GOGAT1and OsNADH-GOGAT2. In nitrogen sufficient field condition, NADH-GOGAT cosuppressed transgenic lines exhibited nitrogen deficient phenotype, such as decreased tiller number, plant height, yeild of per plant and thousand kernel weight. Metabolic analysis showed that suppression of NADH-GOGAT caused a decrese of the contents of leaf soluble sugars, total nitrogen and most of nitrogenous compounds, and an increase of the contents of2OG, isocitrate and free ammounium. The activities of some carbon and nitrogen metabolism related enzymes were also affected. These results proved that NADH-GOGAT playing an important role in rice carbon and nitrogen metabolism, and it is indispensible in the process of improving NUE in rice.2. Constitutive expression of OsICDH2in rice caused cosuppression of both OsICDH1and OsICDH2. Compared with wild type, ICDH activity in leaves and roots of cosuppression lines decreased43.5%-76.6%and19.4%-56.4%, respectively. Under sufficient nitrogen conditions, ICDH cosuppressed plants showed a retarded growth phenotype, yield of per plant in field were significantly reduced, which suggests that ICDH is essential for rice to maintaining the high production.3. When exposed to high temperature for3-6days, ICDH cosuppressed plants become chlorosis and eventurally died, while the wild type grows normally. Biochemical and physiological analysis showed that, under high temperature condition, when compared with wild type, ICDH cosuppressed lines showed decreased contents of leaf chlorophyll, nitrate, some organic acids and solulble sugars, and increased contents of leaf free ammonium, total free amino acids, asparagine and glucose-6-phosphate, increased activities of glutamate dehydrogenase, aspartate aminotransferase, glucose-6-phosphate dehydrogenase, fructokinase and glucokinase, and decreased GS activities, higher expression levels of OsGDHl and OsGDH2, and lower expression levels of OsGS2and OsFd-GOGAT1. We suggest that both NAD-IDHs (NAD-dependent isocitrate dehydrogenase) and cytosolic ICDH can provides2OG for GS-GOGAT cycle for nitrogen assimilation, NAD-IDHs are responsible for the major source of2OG, whereas the2OG produced by cytosolic ICDHs might act as an important compensation, especially in circumstances, such as high temperature, in which the rate of2OG production by mitochondria is inadequate. Glutamate dehydrogenase, aspartate aminotransferase, and2OG translocators may also help to supplement the inadequate supply of2OG under high temperatures.4. Totally65OsAAT genes were identified in rice genome. Among them,49were supported by full length cDNA, some of OsAAT genes exhibited tissue preferential or nitrogen starvation induced expression patterns. T-DNA insertion mutant lines of11OsAAT members were identified and characterized, some of them exhibited modified carbon and nitrogen contens, suggesting these genes play significant roles in carbon and nitrogen distribution in rice.更多还原