Expression and Characterization of Human Superoxide Dismutase Gene in Tomato Plants

• Main Authors: chen pi yu, 陳碧榆
• Other Authors: 江主惠
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/05499192172610673344

碩士 === 大葉大學 === 分子生物科技學系碩士班 === 93 === The extracellular superoxide dismutase (EC-SOD) is predominantly located in extracellular fluids and extracellular matrix of tissues and plays a central role to scavenge O2.- generated in extracellular space of mammals. The objective of this study is to transfer a human ec-sod gene into tomato plants to see if the SOD protein and activity are properly expressed, and then to investigate the responses of the transgenic plants under environmental stress. The human ec-sod gene was constructed and transformed into tomato plants (Lycopersicon esculentum L. cv. Known-You 301). Three different constructs were obtained from PCR amplification and cloning process. The first construct contained a signal peptide at the N-terminus of the sod gene for extracellular secretion. The second construct with no N-terminal signal peptide but with a sequence of KDEL located at the C-terminus of the sod gene for targeting to endoplasmic reticulum (ER). The last construct contained sod gene but with none of the signal peptide. These three constructs were introduced into tomato plants either by Agrobacterium-mediated transformation or by particle gun bombardment. The regeneration of non-transgenic Known-You 301 had the best regeneration rate in the MS medium containing 2 mgL-1 Benzylamino purine (BA) and 0.02 mgL-1 naphthaleneacetic acid (NAA). From the kanamycin sensitivity test on non-transgenic Known-You 301 tomato plants, it was found that 40 mgL-1 of kanamycin was high enough to stop the regeneration of tomato. The transformation results showed that 52 putative EC-SOD transgenic lines were regenerated from 2945 cotyledons, and the transformation rate is 1.5-1.8 %. Forty of them were positive by PCR amplification using the specific primers to nptII gene and to sod gene. The expression of EC-SOD in the putative transgenic lines were confirmed by RT-PCR amplification coupled with SmaI restriction digestion. Western blotting analysis was used to further detect the expression of the sod gene by using the anti-SOD antiserum specific against to the peptide from amino acid residue 221 to 235 of EC-SOD. Two clear protein bands with the molecular weight about 31 and 33 kDa were found in all the transgenic tomato lines. In the NBT activity assays, one extra band was observed in the transgenic plants. This pattern was disappearing after treated the gel with 3 mM KCN. The result suggested that the extra band may be the EC-SOD. Transgenic line 8-2 that contained the N terminal extracellular signal peptide of SOD was further check for the expression of SOD at 37℃. A specific protein about 31 kDa was observed at the second day of the heat treatment and was lasting to the fourth day. The protein band was still present in the transgenic plants three days after the plant moved back to the normal environment.