【作者】 马珂；

【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， 2009， 博士

【摘要】 水稻是世界重要的粮食作物。目前,随着全球耕地面积的不断减少,粮食危机逐渐显现,为了满足粮食需求,人们大量地向农田施入各种化学肥料以保证产量,特别是氮肥,因为氮肥是影响和制约粮食产量的最为重要的因素之一。然而,大量施入氮肥带来了许多负面影响,例如生产成本增加、经济浪费以及环境污染。传统的育种手段已经无法满足人们日益增长的粮食需求。因此,利用分子生物学技术快速高效地获得氮高效吸收和利用的转基因水稻新品种对我国水稻生产甚至是整个农业发展起着十分重要的意义。另外,各种非生物逆境(干旱、冷害和高盐等)对植物尤其是作物的生长和发育也有着非常重要的影响,因此研究和逆境相关的基因对提高作物对干旱、高盐和低温等逆境的耐受能力具有重要意义。本研究通过超量表达低氮诱导表达基因、铵盐转运蛋白基因(AMT)和硝酸盐转运蛋白基因(NR乃,对转基因植株进行表型分析和生理生化研究,以获得氮高效吸收和利用的转基因水稻新品种；通过对C3HC4类型的锌指蛋白家族成员进行生物信息学分析以及胁迫条件下的表达分析,找到一些和逆境相关的锌指蛋白成员。主要结果如下：1.使用生物信息学方法在NCBI数据库搜索获得水稻低氮诱导表达基因、铵盐转运蛋白基因(AMT)和硝酸盐转运蛋白基因(NRT)的序列信息；并通过cDNA文库和PCR扩增获得基因。以载体pCAMBIA1301S为基础,构建超量表达载体,以35S启动子启动基因在水稻中进行组成型大量表达。通过Southern和Northern杂交,对转基因植株进行转基因拷贝数和表达量的检测,来获得超量表达的单拷贝转基因植株。对转基因植株进行水培筛选和田间鉴定,找到和野生型植株表型有差异的转基因植株进行下一步研究。2.用Real-time PCR的方法检测水稻全生育期27个组织和器官中10个铵盐转运子基因(OsAMT1;1,OsAMT1;2, OsAMT1; 3,OsAMT2;1, OsAMT2;2, OsAMT2;3,OsAMT3;1, OsAMT3;2, OsAMT3;3,OsAMT4;1)的表达水平。结果表明：OsAMT1;1, OsAMT1;2, OsAMT2;1和OsAMT4;1在27个组织或器官中广泛表达,属于组成型表达的铵盐转运子,然而另外6个铵盐转运子基因特异地在某些组织或器官中表达如根,幼穗、胚乳等等。3.用Real-time PCR的方法检测氮饥饿以及不同氮源处理条件下铵盐转运子基因的表达变化。结果表明在缺氮条件下6个铵盐转运子(OsAMT1;3,OsAMT2;1, OsAMT2;3,OsAMT3;1,OsAMT3;2,OsAMT4;1)在地上部和地下部中的表达不发生改变,另外在不同的时间点5个基因在地上部或地下部的表达发生变化。另外,经过不同氮源处理后植株根中除了OsAMT1;1外,其他的铵盐转运子基因在转录水平发生改变。这些结果表明大多数的铵盐转运子基因在转录水平上至少能被一种氮源所影响,并且在氮饥饿条件下,一部分铵盐转运子基因的转录水平会快速发生改变使得植株能够适应外界改变的环境。4.利用农杆菌介导的遗传转化的方法将OsAMT2;1和OsAMT2;3这两个基因分别导入到水稻中花11中进行超量表达。经过水培筛选发现,和野生型对照中花11,超表达OsAMT2;1的转基因植株长势瘦弱,鲜重和分蘖数和对照相比分别减少30%,转基因植株根内游离铵含量和总氮含量大约上升了10%。超表达OsAMT2;3的转基因植株同样长势瘦弱,植株的鲜重和分蘖数与对照相比减少了大约30%,转基因植株根内的游离铵含量下降了10%。通过15N的吸收实验同样证明了超表达OsAMT2;3的转基因植株吸收铵的能力大约下降了10-15%。5.水稻中典型的C3HC4类型的锌指蛋白OsRHC一共有29个成员,对它们的基因结构、顺式作用因子、染色体定位、进化关系、以及表达作了系统的分析,并通过Real-time PCR对部分OsRHC基因在激素处理和逆境胁迫条件下的表达变化做出研究。分析结果表明29个OsRHC基因中有5个C3HC4型锌指蛋白成员特异地在生殖器官中表达,在非生物胁迫下12个基因在苗期受到激素或逆境的调节。这些结果对阐述C3HC4型锌指蛋白在植物生长发育和逆境响应中的作用非常有帮助。更多还原

【Abstract】 Rice is the most important crop in the world. At present, the cultivated land in China is decreasing day by day. Various chemical fertilizers especially nitrogen fertilizer were applied into the field in order to satisfy people’s demand for food. As we all know nitrogen is the most important nutrient element for crop production. However, the substantial inputs of nitrogen fertilizer have taken negative effects such as the increased production cost and environment pollution. Obviously, the conventional breeding methods can’t keep up with the increasing food demand of the people. It will play a very important role in rice production and even the entire agricultural development in our country by using transformation method to obtain the new rice variety of high efficiency of nitrogen absorption and untilization. In addition, abiotic stress, such as drought, salt, and low temperature, frequently influences rice growth, development. It becomes more and more urgent to study the genes related to abiotic stress to improve rice tolerance to these abiotic stresses. In this study genes including nitrogen-induced genes, ammonium transpoter(AMT) and nitrate transporter (NRT) were over-expressed in rice plants, the phenotype and metabolic level of transgenic plants were analyzed by molecular biology and physiology methods in order to obtain the transgenic rice plant with the higher efficiency of nitrogen absorption and untilization. The C3HC4-type zinc finger proteins were studied by informatics and the expression level of OsRHCs were analyzed under various stresses. Finally some OsRHCs related to abiotic stress were found. The main results are as follows:1. The sequence information of genes including nitrogen-induced genes, ammonium transpoter(AMT) and nitrate transporter (NRT) were obtained from NCBI database using bioinformatic methods. And the genes were isolated by PCR amplification or cloned from cDNA library in our lab. The pCAMBIA1301S vector which contained 35S promoter were used to overexpress the target gene by an Agrobacterium tumefaciens-mediated transformation method. The copy numbers and expression level of transformed genes in transgenic plants were also checked by Southern and Northern blot respectively. In order to find the differences between the transgenic plants and wild-type plants, the overexpressed transgenic plants with one or two copies were studied in hydroponic culture condition and in field.2. The ammonium transporter (AMT) family contains ten members(OsAMT1;1, OsAMT1;2, OsAMT1;3, OsAMT2;1, OsAMT2;2, OsAMT2;3, OsAMT3;1, OsAMT3;2, OsAMT3;3, OsAMT4;1) in rice (Oryza sativa). Here we systemically examined 27 rice tissues or organs of whole-life time by Real-PCR method. The results showed that OsAMT1;1, OsAMT1;2,OsAMT2;1,OsAMT4;1 were widely expressed in 27 tissues or organs, while other six genes were specifically expressed in some tissues or organs, such as root, panicle, endosperm and so on.3. Expression levels of OsAMT genes with nitrogen deprivation or different nitrogen forms treatments by Real-PCR method. The results showed that among the ten OsAMT genes, the expression levels of five genes (OsAMT1;3,OsAMT2;1, OsAMT2;3, OsAMT3;1, OsAMT3;2 and OsAMT4;1) did not change under the N deprivation both in root and shoot under nitrogen deprivation,and the expression level of the other five genes were up-or down-regulated at different time point. In addition, all genes except OsAMT1;1 were influenced by different nitrogen forms. The results suggested that most ammonium transporters were affected by at least one nitrogen form and could quickly respond to the nitrogen deprivation condition at the transcriptional level to adapt the complicated environment.4.OsAMT2;1 and OsAMT2;3 were overexpressed respectively in rice Zhonghua 11 (Oryza sativa L. ssp japonica) by an Agrobacterium tumefaciens-mediated transformation method. The results showed that overexpression of OsAMT2;1 in rice results in enhanced ammonium uptake but impaired growth under normal nitrogen condition, the tiller number and fresh weight of transgenic plants were about 30% lower than that of control (Zhonghua 11) and the free ammonium in root of transgenic plants were more than 10% higher than that of control(Zhonghua 11), respectively. Similarly, overexpression of OsAMT2;3 in transgenic rice decreases the ammonium uptake and impaired growth. 15N-labeled ammonium uptake experiment was also proven that approximately 10-15% decrease in root uptake capacity for N-labeled ammonium.5. Here, we report the identification and characterization of 29 typical OsRHC genes in rice, describing the gene structure, cis-elements, genome localization, phylogenetic relationship of each member and gene expression. We have used Real-time PCR to perform a characterization of the hormone-regulated and abiotic stress-regulated OsRHC genes. These results help us to understand the function of OsRHC genes for plants’ growth and development and the responses to abiotic stress更多还原