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本研究采用RT-PCR方法克隆得到水稻细胞质抗坏血酸过氧化物酶(OsAPX1)基因全长cDNA(基因登录号:D45423);将该基因构建二元植物表达载体pBI121-OsAPX1;用农杆菌EHA105侵染介导烟草叶盘转化法转基因,获得转基因植株,PCR鉴定遗传转化的烟草植株成功地整合了水稻OsAPX1基因;Northern blot鉴定结果表明转基因T1代植株Ta在mRNA转录水平上表达;对Ta株系进行Kana抗性鉴定,表明转基因T2代株系有抗性;对T2代株系做NaCl、NaHCO3、Na2CO3抗盐性鉴定,结果表明与对照相比表现出转基因植株抗(耐)性提高;取T2代植株的叶片在不同浓度H2O2处理下,抗H2O2毒害的能力显著强于对照,转基因植物的抗性有所提高,有望在抗盐碱性育种上应用。
In this study, the full length cDNA of OsAPX1 gene was cloned by RT-PCR (gene accession number: D45423). The binary vector pBI121-OsAPX1 was constructed and was infected with Agrobacterium tumefaciens EHA105 Transgenic plants were transformed by tobacco leaf disc transformation, and transgenic plants were obtained. Transgenic tobacco plants were identified by PCR and OsAPX1 gene was successfully integrated into rice plants. Northern blot analysis showed that Ta of transgenic T1 plants was expressed at mRNA level. Kana resistance test showed that the transgenic T2 generation lines were resistant; T2 generation lines made NaCl, NaHCO3, Na2CO3 salt resistance identification, the results showed that compared with the control transgenic plants resistant (tolerance) increased; take T2 Under the different concentrations of H2O2 treatment, the plant leaves had significantly higher resistance to H2O2 toxicity than the control plants, and the resistance of transgenic plants was improved, which is expected to be applied in salt-resistant and alkaline breeding.