Agrobacterium-mediated Transformation Of A Dendrobium Orchid With The Aiia And Hacd Gene

Dendrobium is one of the most popular ornamental flowers in the world. However, they're prone to be infected by the soft rot disease, which tends to result in a lot of loss. The pathogenic bacteria called Erwinia carotovora. are responsible for the soft rot disease on Dendrobium and their expression of virulence factors is coordinated by AHL-mediated quorum-sensing system. The AiiA protein encoded by the aiiA gene from Bacillus thuringiensis (Bt) has been reported to inactivate bacterial virulence through degrades signal molecules of quorum-sensing system. Due to this, it will be one of possible strateges to promote resistance to soft rot; Antibacterial peptides were toxic against a broad- spectrum of bacteria, fungi, viruses, protozoa and cancer cells, they even could directly kill bacteria. So, it will also be one of possible strateges to promote resistance to soft rot; In this study, prokaryotic expression vector of fused aiiA-hacD gene was constructed and expressed in E.coli. Den.Tungku.Anis was transformed with Agrobacterium tumefaciens carrying the fusion gene to expect of acquiring plants that will be more resistant to Erwinia carotovora. infection than the wild-type plants.The region of hacD gene from cotton budworm by PCR amplification (Helicoverpa armigera ), which only codes mature antibacterial peptides, were fused with down-stream sequence of the aiiA gene from Bacillus thuringiensis and a factor Xa recognizing site was designed between them. The resulting fusion gene, aiiA-hacD, was cloned to expression vector pQE-30 (Qiagen) and effectively expressed in E.coli M15. The 34-kDa recombinant AiiA-HacD protein was purified and analyzed its biological activity. The bioassay of AiiA-HacD fusion proteins suggested that only HacD released from the fusion protein can inhibit the reproduction of E.coli K12D31. Both the fusion protein and the production of fusion protein digested by factor Xa protease showed AHL-degrading activity. Besides, the production of fusion protein digested by factor Xa protease could increases the ability to inhibit soft rot caused by Erwinia carotovora.The fusion gene, aiiA-hacD, was cloned into the binary vector pCAMBIA1301 to obtain the recombinant plasmid, pC-AH. The fused gene was driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter and stopped by the nopaline synthase terminator(NOS). Plant expression vector pC-AH were transformed into the Agrobacterium strain EHA105 to create the engineering strain contains aiiA-hacD gene. Then, Dendrobium was transformed by EHA105 equipped with pC-AH. After 17months of selection culture, 13 hygromycin-resistant plantlets were acquired. There were 3 transgenic plantlets containing the fused gene after kanamycin screening, PCR detection and Southern blot analysis.