Established Alfalfa Genetic Transformation System Of Antimicrobial Peptide Rev4 Gene By Agrobacterium Tumefaciens

Alfalfa(Medicago. Sativa. L) is known to the "king of grass",it is excellent and the most widely distributed forage around the world. It has long been around two goals about cost-effectively improve the yield and quality of alfalfa made unremitting efforts, traditional breeding methods waste a long of time, the high cost and limited availability of germplasm, plant genetic engineering is more scientific and accurate. Agrobacterium-mediated genetic transformation method is the most studied and one of the most frequently used methods. Indolicidin is a 13 residue peptide amide(IL-PWKWPWWPWRR-NH2), originally isolated from bovine neutrophil cytoplasmic small particles, with broad-spectrum antibacterial activity and antiviral activity. Antimicrobial peptide Rev4 is a reverse design analogs according to Indolicidin.In this experiment, the explants were mature alfalfa seeds, overall seed infection by genetic transformation method by Agrobacterium tumefaciens A.GV3850. First, alfalfa seed after infection by the kanamycin 75 mg / L(Kan75) pressure screening 45 days, get 68 alfalfa plants have kanamycin resistance. Extract the genome of resistant plants, and its analysis of PCR amplification, including PCR detected 40 resistant plants were positive, after the PCR product was recovered by the Huada Beijing Genomics limited company sequenced. The successful sequencing outcome at the National Center for Biotechnology Information NCBI(http://www.ncbi.nlm.nih.gov/) sequence comparison and analysis, we use Nucleotide Blast, successfully identified 13 plants which transfer Rev4 gene. Spray concentration 8mmol / L salicylic acid inducible Rev4 gene expression, by semi-quantitative RT-PCR for gene expression in transgenic plants were analyzed, Confirmed antimicrobial peptide Rev4 gene expression in transgenic plants successfully.In this study, use of Agrobacterium-mediated infection of alfalfa seeds overall method, successfully antimicrobial peptide Rev4 gene into alfalfa, get 13 transgenic lines, including 10 successful expression. The whole process is infecting our laboratory has proprietary patented technology, with high conversion rate, short experimental period, the experimental work less and so on.Antimicrobial peptides turn alfalfa will greatly enhance disease resistance, insect resistance and production, and has great potential and advantages will be in the animal feed industry, transfer alfalfa of antimicrobial peptides Rev4 as feed to clear the harmful viruses, bacteria and fungi of animals, significantly reduce intestinal diseases, improve disease resistance and improve livestock meat. Antimicrobial peptides are non-specific immune response substance, therefore, antimicrobial peptides will greatly reduce the possibility of resistance. Transfer of antimicrobial peptides may develop green alfalfa breeding special feed, which not only has significant ecological benefits, but also has great economic and social benefits.