Clone And Prokaryotic Expression Of Plant Defensins Genes From Cruciferae And Transgene To Arabidopsis

Plant defensins are small, basic peptides that exhibit antimicrobial activity against a broad range of bacteria and fungi. To date, more than 80 different plant defensin genes from different plant species, such as Arabidopsis, Raphanus are isolated. Because of their three-dimensional folding pattern, they can inhibit the grouth of many fugi without damaging plant. Plant defensins can protect plant seeding and fight against pathogens which attack plant.In this study, we designed a pair of primers 5'-ATGGCTAAGTTTG CTTCC-3' and 5'-TTAACATGGGAAATAACAGATAC-3' based on the cDNAs of cruciferae plant defensins from Genebank. Sad and Rsvd genes with the length of 243bp were amplified from Sinapis alba L.and Raphanus sativus vas. Raphanistroides seeds. In comparison with other plant defensins registered in Genebank, Sad has 90% identitiy to Rs-AFP1 and R.S-AFP2 nucleic acid sequence and Rsvd has 98% identitiy to Rs-AFPl, 92% to Rs-AFP2 and 87% to PDF1.1 in Arabidopsis.Multiple biosoftwares are used for bioinformatics analysis. The comparisons indicate that Sad and Rsvd have the following characters: (1) eight cysteine; (2) four disulifide bridges; (3) a signal site between 29 and 30 amino acids;(3) Csap three-dimensional folding pattern.In further study, the cDNA of Sad gene was inserted into the protein expression vector, GTK. The best condition to induce Sad gene expression is incubation of bacteria at 18°C for 6 h with 0.1 mM IPTG. 8 M urea, 50 mM Tris-HCl(pH8.0) and 0.4% 2-ME was used to isolate the fusion protein.Used Pfu DNA polemerse to amplifie Sad gene from pMDl 8-Sad. Insert Sad into pBI121 vector. Transegene to Arabidopsis by in plant method to study Sad function.