| Nowadays, more and more antibiotics have been developed and used tocombat with various infectious diseases that threaten the health of humans,but it has been found that many strains of microorganisms are resistant to the commonly used antibiotics.Therefore,it is a hot focus to find novel kinds of antibacterial drug with great developing potential,such as antimicrobial peptide. Most of antimicrobial peptides are small cationic peptides with characteristics of molecular weight<5kDa,heat-resistant,water-soluble,low immunogenicity and rapid action.Both cationic and anionic peptide antibiotics contribute to the innate host defense against a number of microbial pathogens,such as bacterial,fungal pathogens,virus and parasite, which are resistant to conventional antibiotics.Some of them have anticarcinogenesis without affecting human normal cells.Cecropins were the first peptide antibiotics found in 1972 in the pupae of Cecropia(a giantsilk moth).By now,antibacterial peptides have been known to exist broadly in insects,plants,animals and human body in vivo. Because the antibacterial peptides have antibacterial activity to the procaryotic cell,they are hard to be expressed in engineering bacteria of protocaryon.With the rapid development of molecular biology and biotechnology, the research of the transgenic plant made a series of breakthrough achievements, so that more and more people are beginning to pay attention to it. The application of recombinant transgenic plant in the production ofvaccines, antibodies and pharmaceutical proteins has become a hot point in the plant genetic engineering both at home and broad. Compared to other bioreactor systems, plants allow the cost-effective productionof recombinant proteins on an agricultural scale, while eliminating risks of product contamination with endotoxins or human pathogens.Another advantage of the use of plants in recombinant protein production is that vaccine candidates can be expressed in edible plant organs, allowing them to be administered as unprocessed or partially processed materials.The hybrid peptide CecropinAD (CAD), which is composed of 1-11 residues of Cecropin A and 12-37 residues of Cecropin D, displays higher antibacterial and antitumor activities. CecropinAD (CAD) antibacterial activities and antibacterial spectrum significantly higher than the Cecropin aand Cecropin DTo explore a new approach to express the hybrid peptide CAD in the alfalfa,the cDNA sequence encoding CAD was obtained by recursive PCR(rPCR),and inserted the control elements to improve the expression level in the transgenic plants,such asΩsequence,Kozak sequence and KDEL sequence,and cloned into the vector pCAMBIA1390.This recombinant plasmid was transformed into A.tumefaciens LBA4404 and transferred CAD into alfalfa by Agrobacterium tumefacien-mediated method. And then selected the transgenic alfalfa plants on select culture medium which consist hygromycin at the concentration of 20 mg/L. The results of PCR,Southern blotting and bacteriostatic activity analysis showed that the CAD gene were successfully expressed in alfalfa.detected by the PCR,Southern blot and bacteriostatic activity, the results were summarized as follows:1:the inserted CAD gene was modified with plant preferring codes.2:We inserted the CADgene into p1390R,successfully get the plant expression vector p1390RCAD. 3:DNA PCR and Southern blot results prove that the target gene is successfully integrated into the genome of Alfalfa (Medicago sativa L).4:RT-PCR results indicate that the target gene is transcribed in the transgenic line.5:The results of bacteriostatic activity analysis showed that the CAD gene were successfully expressed in alfalfa.
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