Gene Cloning And Transformation Of Antioxidant Enzymes In The Chloroplast Of Tomato

Reactive oxygen species in plants can be scavenged by antioxidant enzymes (SOD, APX). Superoxide radical photogenerated by Mehler reaction is disproportionated to H2O2 catalyzed by SOD. APX is the key enzyme in scavenging of hydrogen peroxide in chloroplasts. It is essential in protecting chloroplast and other cellular components from photodamage. Two tomato cultivars with different chilling sensitivity were used, and activities of antioxidant enzyme were investigated under chilling stress. The cloning and transformation of APX and SOD from the tomato were done. The main results were as follows:1. Under chilling treatment (4℃), antioxidant enzyme activities in two tomato cultivars were investigated. Under chilling treatment for first 4h, activities of SOD, APX, POD all increased, and reached to the peak on 6h, 6h, 2h, respectively and then decreased. The enzyme activities of chilling-resistant cultivar L402 increased more quickly than chilling-sensitive cultivar Zhongza No.7. However, the activity of POD increased continually under chilling stress.2. Total RNA from functional leaves of L402 treated by chilling stress for 6h was extracted. According to conserved amino acid sequences of APX of other plants, we designed a pair of primers and obtained a cDNA fragment about 405 bp from tomato leaves with RT-PCR. It shared high identity with thylakoid-bound APX genes. It was registered in GenBank(AF413573).3. 5' and 3' end sequences of the APX gene was cloned with 5'and 3' RACE-PCR. Further a full-length cDNA sequence was obtained. Chloroplast Cu/Zn-SOD gene sequence from tomato leaves and a cDNA fragment about 962 bp encoding 217 amino acid were obtained. Analysis and nucleotide BLAST of this sequence showed that it was the target sequence, which shared 82%, 82% and 80% identity with4. published sequence of spinach, bean and Arabidopsis thiana in GenBank, respectively. The target sequence was obtained and registered in GenBank (AF527880).5. Sense APX gene expression vectors under control of 35S promoter were constructed and transformed into tobacco (Nicotiana tabacum L.). PCR and APX activity detection showed that transgenic plants were obtained.