Cloning And Expression Analysis Of Thioredoxin Gene Trxf1 From Tomato

In this study, tomato cultivar ‘shuangfeng 87-5’ was used as experiment material, we cloned the ORFof Trxf1 gene from Tomato leaves by PCR method, successfully constructed the plant expression vectorp BI121-Trxf1 and the prokaryotic expression vector p ET32a-Trxf1, and then we analysised the real-timefluorescence quantitative of Trxf1 gene. The main results obtained were presented as follows:1.Using the coding region of the gene, according to the ORF sequence we predicted amino acidsequence, then we analysised amino acid homology and conducted phylogenetic analysis, tomato andpotato has the highest homology and the model plant Arabidopsis has the lowest homology; Phylogenetictree showed that tomato and potato genetic kinship of Trxf1 gene were closest, Arabidopsis and othercruciferous plants were farthest.2.Using the coding region of the gene and the plant expression vector p BI21, we constructed the plantexpression vector p BI121-Trxf1 successfully. Tomato plants were transformed via Agrobacterium tumefaciens, PCR and RT-PCR analysis indicated that the foreign gene Trxf1 had been integrated into the genomeof tomato and had acquired expression.3.We cloned the ORF of Trxf1 gene from Tomato leaves by PCR method, successfully constructed theProkaryotic expression vector p ET32a-Trxf1, The recombinant plasmid was transformed into E.coliTransetta(DE3) host strain, induced by IPTG and obtained 25 k D(Mw) protein, purified by affinitychromatography and obtained soluble protein Trxf1 fusion protein. With anti-His tag monoclonal antibodypurified protein identified by Western blot. this checked the target protein in our protein. By Protein Gaffinity chromatography purified soluble protein p ET32a-Trxf1, immunizing New Zealand white rabbitswere prepared Trxf1 polyclonal antibody(Trxf1-KLH), ELISA determine antibody titer 1/6400, Westernblot detect antibody specificity, the validity of Trxf1 protein and polyclonal antibodies were confirmed.4.Using real-time quantitative PCR analysis Temporal and Spatial expression of tomato Trxf1 geneunder salt stress, The gene was expressed in different tissues of the tomato, and in the same period, theexpression is highest in leaves but rarely express in roots; In the stems and leaves organs, salt stress theexpression of the gene was reduced significantly under salt stress; In addition, it was analysised thatexogenous Se on genes expression of related thioredoxin systerms in processing tomato seedlings undersalt stress using real-time fluorescence quantitative PCR. Level of gene expression among Fd/Trx systemwere down-regulated under Na Cl stress, compared to Na Cl stress, these genes expression were significantlyup-regulated under treatment of Se+Na Cl..Level of gene expression among NTS system were up-regulatedunder Na Cl stress, and application of exogenous Se alleviated this trend.