Study Of Transformation On Six Ascorbate Peroxidase Genes From Populous Tomentosa Carr. Into Tobacco

The imposition of environmental stress leads to increased production of reactive oxygen species (ROS) in plant cells. Ascorbate peroxidase (APX) is an important enzyme of enzymatic detoxication system in plants. It is a heme-containing protein that plays a central role in scavenging H2O2in higher plants. It has been demonstrated that transgenic plants overexpressing antioxidant enzymes showed enhanced oxidative stress tolerance.In this lab six Ascorbate peroxidase genes from populous tomentosa Carr. was already cloned, for their different location in chromosome of populous tomentosa Carr., they are named APX-ⅩⅥ, APX-57,APX-2-Ⅸ, APX-V24,APX-Ⅱ,APX-Z-Ⅸ. In this paper we cloned the six genes with restriction sites and ligased it into pMD18T vector, then plant expression vectors with fusing GUS gene of the six APX genes were constucted.By agrobacterium mediated method transgenic tobacco was acquired which was cultured on MS medium supplemented with kanamycin concentration of100mg/L.For each gene, at least70lines tobacoo with resistance was acquired. Through PCR the six genes was indentified that had already transformed into tobacco, and for each gene, at least10positive lines was acquired.Southern blot analysis was performed to identify the PCR positive lines. The results indicated that the foreign gene APX (pBI121-APX-ⅩⅥ, pBI121-APX-V24, pBI121-APX-57,pBI121-APX-Ⅱ) had been integrated into the genome of tobacco. The transgenic tobacco's resistance to salt stress and drought stress are to be proven by further experiments.