Expression Of Four Penaeidins Using Rice Seed Bioreactor

The discovery of antibiotics has made great contributions to human health,but with the abuse of antibiotics,problems such as "super bacteria" and water pollution have plagued human beings at the same time.Antibacterial peptides have become a good substitute for antibiotics because of their broad antibacterial spectrum,high thermal stability,no drug resistance,no residue and no easy enrichment.However,in practical production and application,the extraction process of antimicrobial peptides(AMPs)is complex,the production cost is high,and the output is difficult to meet the needs of food,feed processing and aquaculture industries.The purpose of this study is to try to produce antibacterial peptides of prawn cheaply through rice seed plant bioreactor to meet the above needs.By means of bioinformatics,the gene family members of Gliadin,an important storage protein in rice seeds,were mined,and the candidate members were identified.Finally,26 members with Gliadin family domain characteristics were obtained.The structural analysis,expression identification based on transcriptomics,and transient transformation identification of its promoter were carried out,and the promoter sequence Pos G2(Os05t0329350-00 gene promoter)with rice seed tissue specific high transcription potential and the plant expression vector p G2M3301 driving the reporter gene GUS were obtained.The artificial coding gene PEN1234 that can express four antimicrobial peptides simultaneously was designed and synthesized,and its plant expression vector p G2M3301-PEN1234 was successfully constructed.Through Agrobacterium tumefaciens mediated rice genetic transformation,herbicide screening,molecular detection and activity analysis,a transgenic rice line Pen Os017 with high expression was finally obtained.The content of Penaeidin,Pen2-1,Penaeidin 3 and Pen4-1 in its seeds is about 251～300 μg/g*FW,which makes the rice transgenic line have commercial application potential.However,the identification of antibacterial peptide activity of transgenic seeds shows that they can maintain good antibacterial peptide activity under various storage conditions.Even after boiling the seeds in hot water,the shrimp antibacterial peptide in the seeds can still maintain the antibacterial activity of significantly inhibiting the growth of methicillin-resistant Staphylococcus aureus.The results of this study showed that the rice seed bioreactor could be used to express prawn antimicrobial peptides without expensive production costs,and could be directly used as an ideal candidate material for antibiotic free food and feed.