【作者】 赵蓉蓉；

【导师】 邹春琴； 童依平； Jorge Dubcovsky；

【作者基本信息】 中国农业大学 ， 植物营养学， 2014， 博士

【摘要】 全球超过半数的人正在遭受微量元素缺乏的影响,这已经成为全球关注的热点问题。作为提供日常饮食能量超过50%的粮食作物小麦,其籽粒微量元素是发展中国家人体所需微量元素的主要来源。作为一年生的禾本科植物,小麦的衰老过程伴随着蛋白质、微量元素和开花期前的贮存物质从营养器官向发育的籽粒转移的研究领域受到广泛关注。小麦GPC-B1基因是一类与籽粒蛋白质、微量元素含量相关的植物特异性NAC转录因子,被认为参与调控包括植物生长发育、防御响应、非生物胁迫响应和衰老过程。目前为止,外源氮素供应和衰老过程对小麦铁锌吸收和累积的作用机制不是很清楚。本文在土培和营养液培养条件下研究氮素供应对小麦吸收和累积铁锌的影响以及氮素是否对与铁锌螯合相关基因-尼克酰胺合成酶的表达产生影响；利用不同GPC的敲除突变体在盆栽和大田试验条件下,研究了GPC家族同源基因在衰老过程中的作用及其对籽粒铁锌累积的影响,并利用RNA测序技术研究小麦衰老叶片转录组水平的变化,主要结果如下：1.土培条件下,增加氮素供应促进了冬小麦叶片中铁锌的累积,主要表现为根系干重和铁锌含量随着施氮量的增加而减少,而同时氮素供应的增加显著提高了冬小麦叶片干重和铁锌含量；同时,与铁锌螯合有关的尼克酰胺合成酶基因TaNAS3和TaNAS2在新叶中的表达量随着施氮量的增加而增加,TaNAS1在根系中的表达量随着施氮量的增加而上升,说明氮素供应促进了根系对铁锌的吸收同时增加向地上部的运输；2.营养液培养条件下,增加氮素供应提高了冬小麦叶片干重和铁锌含量；同时,根系干重和根系铁锌含量随着施氮量的增加而降低；与铁锌螯合相关的尼克酰胺合成酶基因TaNAS2在叶片中的表达量在高氮处理下显著的增加,TaNAS1在根系中的表达量在高氮处理下最高；3.利用六倍体小麦GPC-1同源基因敲除系研究了GPC-1对衰老进程和铁锌再转移过程的影响。结果表明,GPC-1突变株系延缓衰老进程的同时导致籽粒蛋白质含量和铁锌浓度显著降低,同时7个受到GPC-1调控的目标基因在gpc-1双突变株系和野生型对照系的表达模式有明显差异。同时证明了GPC-A1和GPC-D1同源基因对衰老过程和营养物质的再转移过程存在功能冗余现象；4.利用四倍体小麦GPC同源基因敲除系研究了不同GPC同源基因对衰老进程和铁锌再转移过程的影响,结果表明gpc-al单突变株系表现出明显的衰老延迟现象,验证了GPC-1对衰老过程的影响；同时gpc-a1b2双突变株系与gpc-a1单突变株系衰老表型和gpc-b2单突变体株系与野生型对照系衰老表型相似的现象,说明GPC-B2对小麦衰老和营养物质的转移没有明显的影响,转录组水平测序的结果显示四倍体小麦存在有功能的GPC-A2基因；5.土培培养条件下,比较衰老进程中四倍体小麦GPC突变株系RNA旗叶转录组在拔节期、花后12和22天发生的变化,通过测序片段拼接、比对及功能注释后发现催化酶和转运蛋白在拔节期与花后12天下调表达,然而伴随着灌浆期的进行,花后22天旗叶水解酶表达上调,说明在籽粒灌浆期,蛋白质水解酶活性的提高可以增加蛋白质的分解速率有利于后期籽粒中N的转移；而蛋白转移酶在拔节期和花后12天表达量增加,花后22天表达量又下降；6.受到衰老过程GPC转录因子的影响两个未知功能基因依靠反向遗传学的筛选是基于GPC-RNAi转基因株系在花后不同时间点进行的类似RNA测序试验,得到一系列受到GPC调控的未知功能基因的结果。结果表明其中具有Jacalin-like lectin结构域的Isogroup08662基因筛选结果得到是无义突变；Isogroup10136家族成员中的Isogroup10136-2基因属于小麦D基因组；Isogroup10136-3获得23个潜在目标突变体。更多还原

【Abstract】 Malnutrition has been a global hot-spot problem since over half of the global population is suffering the zinc (Zn), iron (Fe) and vitamin deficiencies. Wheat is the dominant staple food comprised over50%of diet and micronutrients content in the grain and are also the main source for human micronutrients absorption. Nitrogen (N) application has been approved to be an effective practice on Fe and Zn accumulation in wheat grain. Less research has been published on how nitrogen application affects the Fe and Zn uptake and accumulation in wheat and the further mechanism between N supply and nitrogenous chelator related to Fe and Zn uptake and translocation.During the monocarpic senescence of cereals plant like winter wheat, positional cloning of GPC-B1genes showed GPC-B1is a NAC domain transcription factor related to senescence acceleration and grain protein and micronutrients remobilization in wheat which resulted in high grain protein and micronutrients accumulation in wheat grain. In this study, the effects of N application on nicotianamine synthase (NAS) genes expression, Fe and Zn uptake and translocation were conducted under potting and hydroponic culture condition. Meanwhile, we identified function of the orthologous genes of GPC-1and evaluated its effects on senescence, Fe and Zn remobilization processes and candidate genes in wheat through different GPC-1genotype mutants. And the RNA seq between wild type and GPC knockout mutants at three stages of senescence was applied to determine the underlying transcriptional changes associated with these phenotypic traits.The main results are as followed:1. Under potting culture condition, N application enhanced leaves dry weight and their Fe and Zn content; meanwhile, root dry weight and their Fe and Zn content were decreased with increased N application. With increased N supply, the expressions of TaNAS2and TaNAS3in leaves were improved with the nitrogen supply in leaves and the expression of TaNASl was specifically up-regulated in roots. These results indicated that increased N supply enhanced NAS gene expression, which may potentially result in more nicotianamine concentration in roots and/or leaves;2. Under hydroponic culture condition, leaves dry weight and their Fe and Zn content were increased and roots dry weight and their Fe and Zn content were decreased with the increased N level. With increased N levels, TaNAS2expression in leaves was highly up-regulated with increased N supply and the expression of TaNASl in root was up-regulated. The similar NAS genes expression pattern in wheat grown in potting and hydroponic culture, exhibited that the enhancement of NAS genes expression had highly responses to increased N supply.3. The gpc-1mutants were used to identify the effect of GPC-1on grain protein, Fe and Zn remobilization. The results showed that a significant senescence delay in both gpc-al and gpc-dl single mutants and a stronger effect was found in gpc-1double mutant than in the wild type. At maturity, grain protein concentration, Fe and Zn concentrations in all gpc mutants were significantly lower than in wild type while thousand grain weight was unaffected. Seven target genes showed significant differences in expression between gpc-1double mutant and control plants at three different stages. GPC-A1and GPC-D1homoeologues played redundant roles in senescence and nutrient remobilization processes;4. A significant delay in senescence was found in gpc-al single mutant compared with wild type. This result also confirmed that the GPC-1gene played an important role in the initiation and progression of senescence. Since the gpc-al/b2double mutant showed similar senescence traits as gpc-al single mutant, GPC-B2was predicated to play non-functional role in tetraploid wheat. Based on the mRNA sequence analysis and phenotype observed in the field experiment, GPC-A2may have function in senescence process regulation in wheat;5. Under the potting culture condition, RNA sequence analysis was used to compare the difference in the transcriptome of wheat during the senescence at heading stage,12days and22days after anthesis. The results showed that the genes expression related to catalytic enzyme and transporter were down-regulated at heading stage and12days after anthesis. With the onset of wheat senescence, the genes expression related to hydrolase enzyme were highly up-regulated at22days after anthesis, which indicated that grain N accumulation may partly come from protein degradation in flag leaves at grain filling stage;6. Based on transcriptome analysis of GPC-RNAi transgenetic plant, isogroup08662and isogroup10136as plant hormone were up-regulated by GPC. By using TILLING for GPC4XDNA pool generated by tetraploid wheat (Desert durum(?)heat, Kronos),5potential target mutants of isogroup08662were identified. The sequencing results showed that synonymous mutation and systemic error existed in isogroup08662. Among three homologues of isogroup10136, isogroup10136-2belonged to wheat D genome. And23potential mutants of isogroup10136-3were prepared for the precondition for phenotype and gene function characterization.更多还原