【作者】 武玉叶；

【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】 多年生黑麦草是一种重要的冷季型草坪草,近年来全球水分紧缺,土壤盐化、次生盐渍化不断加剧,而种植抗旱、耐盐的草坪草能减少水分蒸发,抑制盐碱地土壤返盐,还可以用含一定盐分的水来灌溉,节省水资源。同时在北方及过渡地带,许多草坪草品种都遭受到不同程度冻害,严重影响草坪景观。本研究的目的是通过基因工程的方法将水稻OsNHX1和拟南芥AtDREB1A基因转入黑麦草,提高其耐盐、耐低温或抗旱能力。 对多年生黑麦草再生及根癌农杆菌介导的转化体系研究发现,6个品种黑麦草种子都能诱导出愈伤组织,通过胚性愈伤组织分化再生植株。但激素及添加浓度对不同品种愈伤组织诱导率及分化率影响显著。黑麦草种子再生植株的最适激素条件是愈伤组织诱导添加9mg/L2,4-D,胚性愈伤组织的诱导添加3mg/L2,4-D,分化过程添加0.1mg/L 2,4-D和0.1mg/L6-BA。生根培养基不添加任何激素。在最适培养条件下筛选出愈伤诱导率和分化率最高的品种TopGun。后在最适再生条件下,对影响根癌农杆菌介导转化效率的两个因素进行了研究,发现在摇菌和共培养过程添加200μM乙酰丁香酮及转化前将部分胚性愈伤组织于含0.4mol/L甘露醇渗透培养基上预培养12h,能提高转化效率达19.39%。 利用建立的根癌农杆菌转化体系将水稻OsNHX1基因、拟南芥AtDREB1A基因转入了多年生黑麦草。 水稻OsNHX1基因转化黑麦草得到4个转基因株系,PCR及Southern blotting鉴定表明,OsNHX1基因成功插入黑麦草基因组中。抗盐性鉴定表明,转基因株系比野生型抗盐能力显著提高,盐胁迫下,转基因株系比野生型积累了较高的Na~+、K~+及Pro含量。 拟南芥AtDREB1A基因转化黑麦草得到的转基因株系与野生型相比,植株生长矮小,叶绿素含量增加,叶色加深。表型发生明显改变。而且转基因株系耐低温能力提高。丙二醛含量测定表明,转基因株系丙二醛含量比对照低,膜系统受伤害程度较轻。 本文利用根癌农杆菌介导的方法将OsNHX1和AtDREB1A基因转入多年生黑麦草,获得了抗盐、耐低温能力明显提高的转基因株系。为草坪草的改良奠定基础,必将加快草坪草的育种进程,带来显著的经济效益和社会效益。更多还原

【Abstract】 Perennial ryegrass {Lolium perenne L.) is one of the most important turfgrass species, Currently, worldwide water supply is waning and the salinity of soil is increasing. To grow on these dry and salty lands, grasses such as turfgrass need to have salt-tolerance. Turfgrass grow in dry area can reduce the vaporization of water and decrease the accumulation of soluble salt on soil surface, thus maintain or even improve the soil condition. Furthermore, salt-tolerant turfgrass can be irrigated with secondly cleansed water containing high salinity. At the same time, each year most of the turfgrass are faced with different kinds of winter injury in transition and northern climate region, which had great effects on turf qulity. The purpose of the paper is to obtain salt-tolerant and freezing- or drought-tolerant perennial ryegrass by over-expression of OsNHX1 and AtDREBlA gene.The protocol of efficient regeneration system of perennial ryegrass was studied at first. Callus formation and regeneration via embryogenic calli were observed in all the six used cultivars seeds. Growth regulators and their concentration supplemented had great effect on callus induction and regeneration of perennial ryegrass. The optimized tissue culture conditions for seeds of perennial ryegrass were supplementation of 9mg/l 2, 4-D in callus induction medium, 3mg/l 2, 4-D in embryogenic calli medium, 0.1mg/l 2, 4-D and 0.1mg/l 6-BA in callus regeneration medium. No growth regulators were used in root induction medium. Under the most optimized conditions, significant difference were found among the cultivars with ’TopGun’ having the highest callus induction and regeneration rates which were 92.6% and 48.9%, respectively. Using the optimized regeneration conditions, two factors affecting transformation efficiency, acetosyringone and mannitol, were determined. Addition of 20μM acetosyringone during the incubation of bacteria and the co-cultivation process and osmotic treatment with 0.4mol/l mannitol for 12h before infection increased transformation efficiency to 19.39%.Using the optimized transformation system, the rice vacuolar Na+/H+ antiporter gene, OsNHXl and Arabidopsis AtDREBlA gene were transferred into perennial ryegrass by Agrobacterium-mediated method.Four resistant lines were obtained by Agrobacerium-mediated transformation of a vacuolar Na+/H+ antiporter OsNHXl gene in rice. PCR and hybridization indicated that the OsNHXl cDNA have been inserted into perennial ryegrass genome. The obtained transgenic ryegrass showed better salt-tolerance. The leaves of transgenic plants accumulated higher contents of Na+, K+ and Pro than those of the control plants.Transgenic plants over-expressing AtDREBlA exhibited dwarf phenotype compared with the wild-type plants. The leaf length and width of transgenic plants were considerably shorter than the wild-type plants. The leaves of transgenic plants showed dark-green in conrast to the wild-type plants. Compared with the wild-type plants, the content of chlorophyll of transgenic plants increased. Transgenic plants over-expressing AtDREBlA displayed high level of tolerance to freezing. The content of MDA in transgenic plants was lower than that of wild-type whichindicated that the damage to the membrane of transgenic plants was lighter than that of wild-type plants.In the paper, OsNHX1and AtDREB1A genes were successfully transferred into perennial ryegrass by Agrobacterium-mediated method, the transgenic plants showed salt- and freezing- tolerance, which can lay good foundations for turfgrass improvement, quicken the breeding process thus provide significant economic and social values.更多还原