【作者】 陈惠；

【导师】 王学臣；

【作者基本信息】 中国农业大学 ， 植物学， 2005， 博士

【摘要】 水稻是重要的粮食作物,其产量严重地受到水资源短缺和土壤盐渍的影响。利用基因工程手段,提高水稻的耐盐、耐旱能力,具有深远的应用前景。本研究将OsNHX1和AtNCED3两个基因,分别转化到旱稻和水稻中;同时选择了诱导型启动子rd29A在水稻中超表达AtNCED3基因,希望得到既耐盐、耐旱、耐低温,又不影响生长发育的水稻新品系。 以旱稻IRAT109品种的成熟胚愈伤组织为受体材料,与含质粒p330I/OsNHX1的农杆菌菌株LBA4404共培养,经筛选培养获得了多个超表达OsNIIX1基因的独立转化株系。PCR检测证明目的基因已整合到旱稻的基因组中。进一步Northern blot分析表明:三个转基因株系的T1和T2代植株中目的基因的mRNA表达水平明显高于野生型。通过对转基因株系表型观察和生理生化分析表明:100mM NaCl处理5天后的恢复生长过程中,转基因植株的生长明显快于野生型:200mMNaCl处理下,转基因植株较野生型出现受害症状的时间大大延迟,说明转基因植株耐盐性得到了提高。NaCl处理下,转基因植株叶片和根中Na~+含量的提高和渗透势的降低,说明转基因植株的Na~+/H~+ antiporter的表达量高于对照,能将过多的Na~+累积于叶片或根细胞的液泡中,从而提高了细胞的渗透吸水能力,维持了细胞溶质的离子平衡。 此外,选择了组成型启动子Ubi-1和诱导型启动子rd29A在水稻中超表达AtNCED3基因,通过农杆菌介导法获得了两个水稻品种和两个旱稻品种的多个转基因株系。对水稻品种中作93转基因植株的ABA含量分析表明:组成型表达AtNCED3基因在正常和渗透或干旱胁迫下都能提高转基因抗性愈伤组织和转基因植株叶片内的ABA含量,而诱导型表达AtNCED3基因只在渗透或干旱胁迫下提高了转基因抗性愈伤组织和转基因植株叶片内的ABA含量。从转基因水稻植株与对照的表型观察、生理测定和抗逆性研究等的结果表明:组成型表达AtNCED3基因的水稻植株长得非常矮小、开花时间大大推后,细胞的分裂和伸长以及生长与发育都受到了抑制,降低了正常条件下的蒸腾速率和气孔导度;然而,诱导型启动子驱动的AtNCED3基因的超表达对转基因水稻的营养生长阶段的生长发育虽有轻微影响,但在干旱、盐胁迫和低温等逆境条件下表现出明显的抗逆性。 本研究得到了具有较高抗盐的转OsNHX1基因旱稻株系,并利用诱导型启动子rd29A在水稻中超表达AtNCED3基因,得到了既抗逆,又不影响生长发育的水稻新品系。为利用转基因技术提高作物的抗逆性奠定了基础,并为深入研究AtNCED3基因的功能和ABA的作用机理提供了有用的材料。更多还原

【Abstract】 Rice is an important crop. However, water shortage and salinity soil have become most serious constraint to rice production and yield stability in many rice-growing areas. Producing drought- and salt-tolerance rice lines by plants gene engineering was the most importent approach in rice breeding at present and in the fuilture. In this study, OsNHX1 and AtNCED3 were transformed into upland rice and lower land rice respectively. To obtain new rice lines with enhancing drought, salt and lower temperature tolerance but without affecting their growth and development, we selected the inducible promoter rd29A to overexpres the AtNCED3 gene in rice.Mature embryo derived calli from IRAT109 cultivar were as starting material and had been co-cultivated with A. tumefaciens LBA4404 habaring plasmid p3301/OsNHX1. Seven independent transgenic lines overexpressing OsNHXl gene were created and the object gene has been integrated in the upland genome by PCR assay. Moreover, Northern bolt analysis showed that the expression levels of OsNHXl in leaves of three independent transgenic plant lines were significantly higher than those of wild type plants. The transgenic plants grew faster than wild type plants during the recovery growth upon the removal of 100 mM NaCl treatment for 5days. Under the 200 mM NaCl treatment, the transgenic plants showed delayed appearance of damage symptoms, suggesting their increased salt tolerance. We found that the Na+ content was higher and the osmotic potential was lower in leaves and roots of transgenic plants than that of wild type plants in the present of 200mM or 50mM NaCl, indicating that transgenic plants had more Na+/H+ antiporter and can compartmentalize Na+ into vacuoles, thus enhancing the ability of cells absorbing water by osmosis and maintaining the cytosol ion balance.Through the mothed of Agrobacterium tumefaciens-mediated transformation, we generated a number of independent transgenic upland rice and lower land rice lines in which the AtNCED3 encoding region is under control of stress-inducible rd29A promoter or of strong constitutive Ubi-1 promoter. On the analysis of ABA content in leaves or calli of transgenic plantsfrom Zhongzuo 93 cultivar, we found that constitutive overexpression of AtNCED3 in rice plants caused a great increase amount of ABA content in both transgenic hygrimacin resistant calli and plant leaves under both normal and drought stress conditions. Nonetheless, inducible overexpression of \heAtNCED3 plants resulted in enhancing ABA levels only under drought stress condition. These demonstrate that the manipulation of ABA biosynthese enzyme genes can elevate the ABA content in plants.According to the phenotype, the assay of physiological traits and abiotic tolerance between the control and transgenic plants, we found that Ubi::AtNCED3 plants result in severe growth retardation, delayed flower, significantly decreased transpiration rate and stomatal conductance, and inhibited cell cleavage and elongation. However, the rd29AvAtNCED3 plants exhibited significant drought, salt and lower temperature tolerance under multi-stress conditions, although slight growth retardation were observed during the vegetative growth stage. These results showed that the rd29Apromoter is quite useful to realize efficient expression patterns of AtNCED3 in rice plants with improved drought-, salt- and lower temperature tolerance by manual manipulation.In this study, we have obtained salt tolerance upland lines and abiotic tolerance rice plants using the inducible promoter rd29A overexpressing AtNCED3 gene. We generated the rd29A::AtNCED3 new rice lines, which not only have multi-stress tolerance characteristics but also grow normally. These works laid the foundation of using plant engineering techniques to enhance abiotic stress tolerance in crops. Meanwhile, we can use the transgenic plants to further study the function of AtNCED3 gene and the mechanism of ABA action.更多还原