【作者】 卓晓蕾；

【导师】 张木清；

【作者基本信息】 福建农林大学 ， 作物栽培学与耕作学， 2007， 硕士

【摘要】 本研究以pmi基因作为筛选标记基因，以甘露糖作为筛选剂，通过基因枪轰击遗传转化甘蔗，并研究其对甘蔗糖代谢的影响，同时通过转基因甘蔗的田间种植，评价其安全性。研究结果表明：在基因枪介导的甘蔗福农95-1702遗传转化筛选体系中，pmi／甘露糖筛选体系的甘露糖与蔗糖的配比为7：3为最适合碳源配比，转化效率达7.4％；经过PCR、RT-PCR、Northern杂交等分子检测证实pmi基因已整合到甘蔗基因组，并测得蛋白表达量；转基因甘蔗叶片的的糖代谢分析结果表明，转基因甘蔗己糖激酶的酶活性在甘蔗不同生育期均表现显著上升，丙酮酸激酶的酶活性均显著下降，而蔗糖合成途径的三个关键酶酶活性只在甘蔗的成熟期表现显著的变化，说明pmi基因影响甘蔗糖代谢相关酶类的酶活性；而土壤微生物平板培养和土壤酶活性分析结果显示，转基因甘蔗对土壤环境包括土壤微生物总量和土壤的主要酶类不产生显著的影响。综合以上分析结果，认为pmi基因用于甘蔗的遗传转化是可行的、对环境是安全的，对甘蔗自身生长和糖代谢也没有明显不利影响。更多还原

【Abstract】 Abstract: A promising protocol of obtaining transgenic sugarcane by gene gun was developed in which selective system based on the Escherichia coli phosphomannose isomerase(pmi) gene as selective gene and mannose as selective agent. In this system transgenic sugarcanes were selected on MS medium with mannose(7g/L) and sucrose(3g/L). The transformation frequency was 7．4%. The integration and expression of the transgene was confirmed by PCR, RT-PCR, Northern blot analysis as well as the activity of PMI assayed with the chlorophenol red assay indirectly and quantified directly via spectrispram. Transgenic sugarcanes were planted in the field for risk assessment of pmi gene. The results were shared as fellow the transgenic clones inherited pmi gene. The activity of key enzymes in the sucrose metabolize were affected seriously . In different growth stage of sugarcane, hexokinase increased remarkable and pyruvate kinase decreased remarkable, but the activity of three key enzymes in sucrose synthesis only have remarkable change at the maturity stage. Transgenic sugarcanes do not have remarkable influence to the soil environment including the total quantity of microorganisms and activity of main soil enzymes. It suggests the pmi gene would be safe to the acceptor and the environment.更多还原