【作者】 张红梅；

【导师】 杨振明；

【作者基本信息】 吉林大学 ， 植物学， 2011， 博士

【摘要】 不良的耕作方式和越来越严重的酸雨现象,使酸性土壤的酸化程度和面积在不断加剧和扩大。在酸性土壤上,限制植物生长发育的主要因素之一为铝(Aluminum, Al)的毒害。Al毒主要抑制植物的根系生长,从而影响水分及养分吸收,最终影响植物生长和发育。不同植物种类间以及同一植物的不同基因型间的耐Al性存在很大差异,并且抗Al特性是可遗传性状。利用生物技术手段改良和提高植物耐Al性,培育出适应酸性Al毒土壤的植物品种或基因型,是解决Al毒害问题的经济有效的方法,其前提是明确植物耐Al毒的生理生化及分子机理。从目前世界范围对植物耐Al机制的研究成果看,从生理角度对植物耐Al机制的研究较为透彻,而关于植物耐Al的分子机制研究仍处于起步阶段。利用基因芯片技术研究Al胁迫大豆基因差异表达,从而得到Al胁迫植物基因表达变化的模型,从整体上研究Al胁迫大豆基因的表达变化对分子水平上阐明植物耐Al机制有重要的意义。本研究用Affymetrix大豆全基因组表达谱芯片对Al胁迫大豆根尖与正常培养大豆根尖的基因表达变化进行了分析,获得了完整的Al胁迫大豆根尖差异表达基因数据库。经单因子方差分析(p<0.05),Al胁迫大豆根尖与正常培养大豆根尖基因表达相比,转录水平表达差异2倍以上的基因共639个,其中上调表达基因561个,下调表达基因78个。参考NCBI数据库和Affymetrix Ontology数据库对639个差异表达的基因按照生物学功能分类,发现只有334个基因(52.2 %)有明确的功能注释,305(47.8%)个基因是尚未鉴定的。这334个基因可以分为9个生物学功能组:细胞壁相关基因(5.9%)、激素与信号转导(3.4%)、激酶与磷酸化(6.3%)、初生与次生物质代谢(5.6%)、氧化胁迫(7.2%)、防御反应(7.1%)、转录因子(9.2%)、转运体(4.2%)和蛋白质代谢(3.3%)。利用实时荧光定量PCR方法验证了芯片数据的可靠性。利用实时荧光定量PCR方法分析了Al胁迫下GmFDR3、GmABC、GmSTOP1、GmRNF12和GmWRKY57基因在耐Al基因型大豆吉育70和Al敏感基因型吉育62中的转录水平表达变化。GmFDR3、GmABC、GmRNF12、GmWRKY57在大豆两个品种中不同程度的受Al诱导表达,而GmSTOP1不受Al诱导表达。而GmSTOP1基因在大豆两个品种的表达量差异不明显。通过RT-PCR方法从大豆根尖中克隆到GmRNF12和GmFDR3基因的cDNA全长。GmRNF12编码一个由236个氨基酸残基组成的锌指蛋白家族成员。GmFDR3编码一个由589个氨基酸残基组成的MATE家族蛋白,具有典型的跨膜结构域,进化树分析表明GmFDR3与白羽扇豆LaFDR3的亲缘关系最近。GmRNF12基因的表达受干旱、高盐、低温、SA及ABA的诱导。GmFDR3受SA诱导表达。在Al胁迫下,GmRNF12和GmFDR3基因在大豆根、茎、叶中均有表达,表达量的高低顺序为:根>叶>茎。构建了GmRNF12和GmFDR3两个基因的超表达载体,以农杆菌介导法转化拟南芥,经除草剂Basta筛选得到T1代转基因拟南芥。对转GmRNF12基因T3代拟南芥进行Al胁迫、低温、高盐胁迫处理,对转GmFDR3基因T3代拟南芥进行Al处理,初步结果表明:与野生型拟南芥相比,转GmRNF12拟南芥提高了抗低温、耐盐能力,转GmRNF12和转GmFDR3基因拟南芥耐Al性明显提高。更多还原

【Abstract】 Soil acidification is an increasing problem in the world because of acid rain, intensive agriculture and with the excessive use of nitrogen fertilizers. Al toxicity is considered to be one of the most important limiting factors of crop production on acid soils. Under acidic conditions, toxic forms of Al will mobilized into soil solution, and rapidly inhibit plant root growth, subsequently decrease nutrient and water uptake, and result in poor crop growth and productivity. The ability to resist Al stress is different in genotypic differences among plant species or cultivars within the same species and the ability to resist Al stress is genetic. The identification and characterization of Al resistance genes will not only greatly advance our understanding of the mechanistic functioning of these processes, but,more importantly, will be the source of new molecular resources that researchers will use to develop improved crops better suited for cultivation on the acid soils that comprise such a large fraction of the world’s lands.Using genomic microarray technique researched genes differential expression under Al stress in soybean and obtained a primary gene expression profiling and to undersand the Al resistance mechanism at global transaiptional level.The present study provided the genomic microarray analysis of soybean transcriptional responses under Al stress, in which the genomic transcriptional levels of Al-stressed soybean were analyzed using the Affymetrix porcine GeneChip. Total RNA was extracted from soybean tips collected before and after Al. Results showed that 639 genes were confirmed as having a greater than twofold altered expression, with 561up-regulated and 78down-regulated. 334 genes involved in a wide range of functions, but 305 genes (47.8% )are unknown functions .The functions of the 639 genes were observed by the Gene Ontology tool, available from the Affymetrix web.These clustered into 9 functional groups: cell wall related(5.9%), signal transduction(3.4%), kinase and phosphorylation(6.3%), Primary and secondary metabolism (5.6%),Oxidative reponse(7.2%), Resistance reponse(7.1%), transcription factor(9.2%),Transporter(4.2%),.Quantitative real-time PCR was used to verify the microarray data.Quantitative real-time PCR method detected the differential expression of some genes at different times under Al stress in jiyu70 and jiyu62. The results compared the difference of gene expression characteristics between in jiyu70 and jiyu62.By using RT-PCR technique, GmRNF12和GmFDR3 were isolated from soybean tips. The results of sequence functionally annotated by Blast in NCBI indicate GmRNF12 encodes a Zn-finger type transcription factor consisting of 236 amino acids. GmFDR3 encodes a MATE family protein consisting of 589 amino acids. Analysis of phylogenetic tree indicated that GmFDR3 is more closely related with LaFDR3.Real-time PCR technique was applied to clarify the express pattens response tovarious abiotic stresses about GmRNF12. GmRNF12 was induced by drought,salt , low-temperature ,SA and ABA . GmFDR3 was induced by SA.Two overexpression vectors for two genes were construced and transfornled into Arabidopsis by Agrobacterium-mediated transformation. Identification of Al resistance indicated that the T3 generation of transgenic Arabidopsis plants showed increasing resistance to Al.更多还原