【作者】 张莉；

【导师】 徐江；

【作者基本信息】 中国农业科学院 ， 作物（专业学位）， 2016， 硕士

【摘要】 氮元素是水稻在生长发育过程中需求量最大的营养元素,如何提高作物的氮素利用效率一直是农业科学家们关心的问题。如今,随着分子生物学和转基因技术的快速发展,分子育种已经成为我国农业可持续发展的一个方向。利用转基因技术培育氮高效的水稻新品种的首要任务就是要挖掘出控制水稻氮吸收利用的基因。目前,虽然研究者们已经对氮代谢途径中的一些关键基因进行了克隆和功能分析,但是植物对氮素吸收利用的信号调控网络复杂,相关基因家族成员众多,使得新基因及相关突变体的筛选和功能分析还需大量的工作。因此本研究从筛选具有氮素营养相关表型的突变体着手,明确其生理特性,为后续挖掘控制氮吸收利用的新基因和功能分析奠定基础。主要研究结果如下:本研究通过田间表型调查,从本课题组利用甲基磺酸乙酯（ethylmethane sulfonate,EMS）诱变的227个水稻突变体（遗传背景为粳稻日本晴（Oryza sativa L.spp.japonica））中初步筛选了6个氮素相关的水稻突变体（分别为m6、m7、m8、m17、m18与m47）,进而结合水培的方法,从表型分析、体内全氮含量、氮素生理利用效率、氮代谢关键酶活性等生理方面对这6个氮相关的水稻突变体展开进一步的验证,同时分别构建了突变体与籼稻Dular（Oryza sativa L.spp.indica）和粳稻日本晴的杂交F₂代群体,对突变性状进行遗传分析。经生理鉴定,初筛的6个氮相关的水稻突变体m6、m7、m8、m17、m18与m47均为氮素营养相关水稻突变体。其中,m6是一个氮素吸收缺陷型的水稻突变体;m47是一个氮利用缺陷型的水稻突变体;m7、m8、m17与m18是四个氮相关的叶黄突变体。与野生型（wild type,WT）相比,六个突变体对氮素吸收同化的减弱导致突变体地上部和地下部的总氮积累量显著减少。其中,突变体m6根系对氮源离子（尤其是对NH₄⁺的吸收）的净吸收速率减弱。由于m6的氮素吸收过程受阻,其根系中的氮素浓度低于WT,同化NH₄⁺的谷氨酰胺合成酶（glutamine synthetase,GS）活性降低。六个突变体株系氮吸收同化的减弱导致其地上部生长发育迟缓,干物质积累受阻,因此随着植株的生长,其体内的氮素浓度有所升高,在开花期表现出地上部的氮素浓度高于野生型。六个突变体地上部氮素浓度的提高,使得地上部氮代谢相关的硝酸还原酶（nitrate reductase,NR）和GS酶活性升高。开花期六个突变体植株剑叶中的叶绿素a、叶绿素b和叶绿素总含量较WT有所降低,叶绿素含量的减少又导致了光合速率的下降和碳同化能力的减弱,进一步抑制了光合产物的积累,使突变体表现出株高降低,叶片发黄,生长受阻等表型。遗传分析结果表明m6、m17和m18是单基因隐性突变体,m8是双基因隐性突变体。综上所述,通过对初筛的6株水稻突变体的生理特性分析,本研究鉴定到这6株突变体是与氮素营养相关的水稻突变体,为后续氮素吸收同化相关基因的克隆和功能研究奠定了基础,同时也为探索氮高效的栽培技术途径提供了理论支持。更多还原

【Abstract】 Nitrogen is the maximum-consuming nutrition element in the growth and development of rice,and it is the concerns that how to raise the nitrogen use efficiency of crops.At present,with the fast development of molecular biology and transgenic technology,molecular breeding has become a direction of the sustainable development of agriculture in our country.Prior to develop new efficient breeds using transgenic technology,our first task is to clone the genes that control the nitrogen absorption and utilization efficiently.At present,although the researchers have cloned some key genes in nitrogen metabolism and analyzed their function.But the signal transduction network of nitrogen absorption and utilization in plants is complex,and there are so many members in the famlily of related genes.Screening on new genes and mutants that relate to nitrogen still needs a lot of work,so our study started from screening on nitrogen related mutants that have specific phenotypes and clarified their physiological characteristics and lay the foundation for subsequent new gene cloning and their function researches.The results of the study are as follows:In this study,according to the phenotype of mutants in the field,we preliminarily selected six suspected nitrogen related rice mutants,i.e.,m6,m7,m8,m17,m18 and m47 from 227 EMS induced rice mutants whose genetic background is Nipponbare（Oryza sativa L.spp.japonica）.Then combining with the method of hydroponics,we analyzed the phenotype,total nitrogen assimilation and concentration,key enzyme activities in nitrogen metabolism of the six nitrogen related rice mutants and their wild type plants.At the same time,we hybridized the mutants and Dular（Oryza sativa L.spp.indica）or Nipponbare and constructed the F₂ generation hybirds to do the genetic analysis.Through the physiological analysis,all the six rice mutants including m6,m7,m8,m17,m18 and m47 are nitrogen related rice mutants.m6 is a rice mutant with nitrogen uptake defective and m47 is a rice mutant with nitrogen use defective.m7,m8,m17 and m18are also four nitrogen related rice mutant with yellow leaves.Compared with WT（wild type）,the capacity of nitrogen absorption and assimilation in the six mutants become weaker,resulting that the accumulation of total nitrogen in roots and shootssignificantly reduced.And among them the absorption rate of m6 root system to nitrogen source ions（NH₄⁺and NO₃^-）reduced,especially the absorption of NH₄⁺.Due to the uptake of nitrogen was retarded in roots of m6,the nitrogen concentration was lower in its roots,and making GS（glutamine synthetase）activity lower.The weakened nitrogen absorption and assimilation in the six mutants futher lead to the retarded growth and development,so with the growth of the plant,the nitrogen concentration of shoots increased,and it was higher than that of WT in flowering stage,thus making the activities of NR（nitrate reductase）and GS higher in the shoots of mutants.And the reduction of total nitrogen content in the six mutants may affect the systhesis of chlorophyll,thus making the contents of chlorophyll in leaves of mutants reduced,and the reduction of chlorophyll content directly makes the net photosynthetic rate lower and the capacity of carbon assimilation decline.This restrained the accumulation of photosynthetic products,and leading the plant height of mutants reduced,leaves chlorosis and other phenotypes of mutants.Genetic analysis revealed that a single nuclear-encoded recessive gene is responsible for the mutation ofm6,m17 and m18.Mutant m8 is controlled by two recessive genes.In summary,we screened out six rice mutants associated with nitrogen metabolism through the physiological and genetic analysis.This study will lay the foundation for subsequent gene cloning and their function researches and provide theoretical support for the exploration of technological approaches to economize nitrogen fertilizer in production.更多还原