【作者】 孟宪平；

【导师】 李彦舫；

【作者基本信息】 吉林大学 ， 作物遗传育种， 2007， 硕士

【摘要】 干旱化趋势已成为全球关注的问题,也是困扰我国农牧业可持续发展的重要因素之一。为了改善环境,使干旱、半干旱的土地资源恢复生产能力,用现代生物工程技术培育抗旱植物品种具有重要的意义。野大麦(Hordeum brevisubulatum (Trin.)Link)是性状稳定、生产性能高、适应性强的优良牧草,既能有效改善土壤养分状况,又具有很好的经济价值。根据植物细胞全能性的理论,本研究运用基因工程技术克隆酿酒酵母(Saccharomy cescerevisiae Hansen)的海藻糖-6-磷酸合成酶基因(TPS1),成功构建了含TPS1基因的植物高效表达载体pCBTPS1,并通过根癌农杆菌介导转化到野大麦愈伤组织中。利用组培技术获得7棵转基因野大麦。经过分子鉴定检测,证明目的基因己经整合到野大麦基因组DNA中并在转录水平大量表达。通过形态观察及相关生理生化指标的检测,表明转基因野大麦株系的抗旱性能相对野生型对照有所提高。更多还原

【Abstract】 Drought-stress detriment is the biggest natural disaster on the global, which influences the growing of plants and restricts the formation of the earth`s surface vegetation. Soil structure and earth`s surface plants were broken, which causes land to become desert continuously, and also lowers the use value of land. Those aggravat our living environment increasingly. large-area aridity in some region in our country year in year out influence the development of agriculture and pasture to some degree. Because of the intensify of worsing desert and shortage of fresh water resource, the degree and areas of aridity increases. So breeding new fuchsin of good anti-drought plant to adapt drought environmental condition in the locality is an economic and valid route to reduce dry region. It`s importantly meaningful to research transgenic anti-drought plant.Trehalose is non-reducibility disaccharide generally existing in the natural world,which is combined by two glucose molecule with a, a-1, 1 glucosidic bond. Trehalose has the extremely fervent fuaction to stable protein and structure of cell envelope, which makes organism have important effectiveness of anti-drought, anti-cold, anti-hightemperature and anti-salt-alkali for organism to resist adversity. Many microbes which contain trehalose synthetase system have different mechanism to synthetize trehalose. The substrate to be utilized mainly is some carbohydrate including maltose、cane sugar、dextrin and starch and so on. Trehalose in yeast cell is synthetized in charge of TPS1 , TPS2 , TPS3 and TSLl etc.four genes, which encode polypeptide to compose a trehalose-synthase Complex. TPS1 gene encode trehalose-6-phosphate synthase depended by UDP-glucose. Some experiment confirm only TPS1 gene can make organism obtain ability to produce trehalose. Bionomics of trehalose possesses potential utilize value in improving the antireversion force of plants. Consequently importing TPS1 gene into vegetation and expressing in it to synthetize trehalose expect to raise drought resistance of plants. Hordeum brevisubulatium(Trin.)link is a kind of superordinary perennial forage grass belonging to gramineous type barley genus, alias Wild barley.Generally distribute at northeast、NeiMeng、QingHai and XinJiang etc. in China. Its herbaceous is soft, and kinds of domestic animal like to eat. It has high nutritive value and high production. Meanwhile it can be planted on low-wet land and can set up artificial grassland. It possesses favourable performance and high economic value. Our study takes the Wild barley on steppe in QianGuo country in JiLin province to be recipient materials. According to the theories of totipote of vegetal cell and taking trehalose improving creature antireversion force to be foundation, we carry out the relative research by making use of genetic engineering method and tissue culture technology in order to raise drought-resistance of Wild barley.In our research we first extracted total RNA of Saccharomyces cerevisia, then taking it as template we cloned the trehalose-6-phosphate synthase gene(TPSI) which is 1.507kb long fragment by RT-PCR. Through the same BamH I和Kpn I double enzyme-cut we connected the fragment with pMD18-T vector plasmid, and transferred the plasmid into Agrobacterium tumefaciens DH5α, then screened recon through blue-white spot method. Identification of enzyme-cut and PCR indicated that TPS1 had been cloned into vector plasmid. We sent the cloned sequence to sequencing and the result indictated TPS1 had been cloned successfully. Through the same way we connected TPS1 with the vector which contain Ubi promoter. Vegetation high performance expression vector pCBTPS1 was constructed successfully through Identification of enzyme-cut and PCR. After establishment of Hyg resistance bolting system and tissue culture system which fit to mature embryo callus to induce and differentiat, we carry out genetic transformation that recombined vector was imported into Hordeum brevisubulatum(Trin.)link callus by bacillus mediating. We obtained 24 resistance plants through continuous resistance bolting. Determination result of PCR displayed 7 regeneration plants showed masculine, and the same result as detection of PCR-Southern of the selected 3 plants from those. These proved that TPS1 has been integrated into nuclear genome of Hordeum brevisubulatum. Method of HPLC detected the trehalose content in 2 transgenic plants respectively is 2.226 mg/g and 2.236 mg/g, while the contrast detected very microcrystalline trehalose.In drought-resistance experiment of the transgenic plants, we observed the transgenic plants was influenced less than the contrast did obviously under the same drought stress. Meanwhile appearance of the transgenic plants happened to change such as abnormal growth. Selecting the transgenic Wild barley which showed masculine by PCR-Southern hybridization, we detected Proline contect and chlorophyll contect etc. related drought-resistance index and discovered that expression of TPS1 in vegetation degrades damage degree of cell membrane under adversity condition, also be helpful for accumulation of carbohydrate、Proline and chlorophyll. At present the research of Trehalose mostly take microorganism、animal and seaweed as experiment subject, while the study that trehalose-6-phosphate synthase gene is transformed into Hordeum brevisubulatum(Trin.)link has not been reported. The experiment result showed drought resistance of the transgenic Hordeum brevisubulatum(Trin.)link is strengthened than the wild type.更多还原