【作者】 丁宝建；

【导师】 姜廷波；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2008， 硕士

【摘要】 铁蛋白是一种大的蛋白质外壳,能以一种安全、无毒的形式储存多达4500个铁原子,广泛存在于动植物中。本研究以烟草为受体,以两个烟草铁蛋白基因(NtFer1 andNtFer2,GenBank accession number:AY083924,AY141105)为目标基因,构建铁蛋白表达增强与表达抑制的植物表达载体,用农杆菌介导法转化烟草(SR-1 strain),研究铁蛋白基因表达增强与沉默的效应,从而研究铁蛋白基因的功能,为其在植物基因工程育种中的应用提供依据。用RT-PCR方法从烟草(Nicotiana tabacum)中克隆出两个铁蛋白基因,将其亚克隆到植物表达载体pBI121中,构建两个铁蛋白基因的正义和反义表达载体。利用农杆菌(Agrobacterium tumefaciens)介导法将正义反义铁蛋白基因导入烟草基因组,培育转基因烟草。对转基因烟草T1代植株的卡那霉素抗性分析表明,多数转基因植株后代表现为3:1的分离比例,少数转基因植株的抗性分离表现为15:1或不符合孟德尔遗传分离比例。说明整合到烟草基因组内的外源基因多为单拷贝基因,少数为多拷贝基因。对T1代植株进行PCR检测证明:两个铁蛋白基因的正义反义序列已经整合到烟草基因组中,并且可以通过有性生殖传递给后代。Northern杂交分析表明正义表达铁蛋白基因不同程度上提高了铁蛋白基因mRNA的含量,而反义表达铁蛋白基因在不同程度上降低了铁蛋白基因mRNA的含量。分析四个转基因群体T1代在高浓度铁离子和低浓度铁离子胁迫条件下的株高、鲜重、叶绿素含量、相对总铁含量、超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性和净光合速率等生长性状和生理特性的变化情况表明:过量表达铁蛋白基因从总体上提高了植株在高铁浓度和低铁浓度下的表现;而反义表达铁蛋白基因则降低了植株对高铁浓度和低铁浓度的耐受能力。采用细菌双杂交分析,检测到烟草体内两个铁蛋白之间存在强相互作用,推测这可能是导致两个铁蛋白基因同时被上调节表达和下调表达的原因之一。更多还原

【Abstract】 Ferritin is a kind of large protein shell, that can store up to 4500 iron atms in a safe, bio-avilability form. It is distributed widely in plant and animals. In order to investigate the effects of ferritin gene expression suppression and provide references data for ferritin gene application in plant stress resistance breeding and phytoremediation, the genes encoding tobacco (Nicotiana tabacum) ferritin NtFer1 and NtFer2 (GenBank accession number: AY083924, AY141105) were amplified by RT-PCR and ligated into the plant expression vector pBI121 in sense and antisense orientation in place of the GUS gene. Four groups of tobacco strains (sense NtFer1, antisense NtFer1, and sense NtFer2, antisense NtFer2) were obtained by Agrobacterium-mediated transformation. The seeds of transgenic plants were subjected to Kanamycin screening and most of the strains showed a segregation rate (resistant: sensitive) of 3:1, and a few of them have a rate of 15:1 or not fit to the Mendelian segregation rate. This indicated the exogenous genes had been integrated into the genome of tobacco in single- or multi-copy pattern. The PCR analysis of T1 generation plant also conformed the transgenes can be inherited from T0 plants to T1 plants. Northern blot analysis indicated that the sense expression of ferritin genes can increase the mRNA content of ferritin genes in different extent, and the antisense expression of ferritin genes can decrease the mRNA concentration of ferritin in various degree.The growth and physiological characters of transgenic tobaccos in high- and low-concentration of iron stress condition were studied. The results showed that: under different iron concentration, the activity of POD and SOD, the relative total iron content, concentration of chlorophyll and net photosynthetic rate were increased in the strains expressing sense ferritin genes, whereas, in the case of antisense expression of ferritin genes, these traits were decreased. Enhanced growth of transgenic tobaccos expressing sense ferritin genes were observed at the early development stages, resulting in plant height and fresh weight significant greater than those of antisense-transformants. These results demonstrated that the ferritin genes have positive roles in plant for the tolerance of iron stress condition.BacterioMatch two-hybrid assay indicated the existance of strong interaction in vivo between the two ferritins of tobacco. This result maybe one of the reasons that can be used to elucidate the phenomenon of overexpression or suppression of one ferritin gene can lead to the overexpression or suppression of another ferritin gene.更多还原