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Expression of Cecropin AD in Transgenic Alfalfa

Author: ZhuHaiLin
Tutor: LiXiao
School: Jilin Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: Transgenic alfalfa Antimicrobial peptides Cecropin AD Plant Bioreactor Vector
CLC: Q943.2
Type: Master's thesis
Year: 2011
Downloads: 44
Quote: 0
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Abstract


Recent years due to extensive use of antibiotics, resulting in an increasing number of clinical drug-resistant strains, the search for new types of antimicrobial drugs become a research hotspot. Peptide thermal stability, good water solubility, low immunogenicity, fast-acting, etc., not only can effectively kill bacteria, fungi, viruses and parasites, and there is still a certain anti-cancer effects, and easy to produce drug resistance, eukaryotic cells and low cytotoxicity. With molecular biology and biotechnology, the rapid development of transgenic plants made a series of breakthrough achievements, become more and more world attention. With plant bioreactor production of recombinant vaccines, recombinant antibodies and other therapeutic proteins and abroad has become one of the hotspots of genetic engineering research. Plants can be ruled out as a reactor product is endotoxin or human pathogens risk of pollution, in the expression of proteins can not processed or partially processed after the adoption. Therefore, the plant bioreactor for the production of recombinant proteins provides a broad prospect. Cecropin AD (CecropinAD, CAD) is the cecropin A synthetic N-terminal peptide 1 to 11 C-terminus of cecropin D and 12 to 37 composed of a hybrid peptide peptide having high antibacterial activity and anti-tumor activity . Low levels of peptide-based natural medicine and its important role in this study, through genetic engineering means, in accordance with the expression of alfalfa plant codon preference synthetic cecropin AD, to explore the use of alfalfa as a reactor producing antimicrobial peptide feasibility study for the antimicrobial peptide transgenic alfalfa lay the foundation for the promotion of industrialization. Inserted into the expression vector of exogenous gene expression has a certain role in promoting regulatory elements: Q sequence, Kozak sequence and KDEL tetrapeptide sequence; cloned into pCAMBIA1390 to construct a pCAMBIA1390CAD plant expression vector. The recombinant plasmids were transformed into Agrobacterium tumefaciens LBA4404, and the method by Agrobacterium-mediated transformation into a CAD alfalfa. Containing hygromycin (20mg / L) filtering medium screening transgenic plants by PCR, Southern blotting and antibacterial activity was detected, the following conclusions: 1. Successfully synthesized plant codon preference CAD genes. 2 would be sequenced properly inserted into pCAMBIA1390 CAD gene, and the resulting CAD gene containing plant expression vector pCAMBIA1390CAD. 3.PCR and Southern hybridization showed that the gene has been successfully integrated into the alfalfa genome. 4. RT-PCR results showed that the target gene in various strains of alfalfa in both transcription. 5 antibacterial activity, and the results show that CAD gene has been transferred to alfalfa, and successfully be expressed.

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CLC: > Biological Sciences > Botany > Plant Cell Genetics > Plant Genetic Engineering
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