Transformation To Maize With VP1 Gene Of FMDV O And Asia1 Strain

Foot and mouth disease(FMD) is the most serious infectious disease of livestock in the world.Presently,the traditional inactivated vaccine is widely used for preventing foot and mouth disease.A new outbreak of the disease may have been caused by the virus incompletely inactivated or escaped from the vaccine.With the development of plant genetic engineering,transgenic plants have the potencial to become the cartier of the antigen protein.In comparison with the traditional method,this maize expression system for vaccine expression is more safe and economical.In this study,the antigen gene VP1 of Foot and mouth disease virus(FMDV) O and Asial strain was integrated into the genome of maize inbred line "18-599W" and "416051" throgh genegun and agrobacteria mediated transformation.Afterwards,DNA was extracted from the leaf of the regenerated seedlings. Based on PCR detection,the positive T₁ transgenic lines was self-crossed to get the homozygese T₂ generation for the development of the transgenic maize vaccine of FMDV.In this study,we choose the immature embryo of two maize inbred lines as the explant due to the established high efficiency transgenic system mediated by genegun and agrobacterium.The plant expression vector of the FMDV antigen protein VP1 gene was constructed and the transformation system was also optimized.Specific results were as follows:(1) According to the preference of maize codon,FMDV O strain VP1 gene DNA sequence,Asia1 type FMDV VP1 gene DNA sequence,1095bp Kozak-LTB-linker-Asia/VP1 and 1101 bp Kozak-CTB-linker-O/VP1 gene synthesized by E.coli sensitive enterotoxin B subunit(LTB) and Vibrio cholera toxin B subunit(CTB) respectively were optimized.Aferwards,plant expression vector pC1300-LTB-Asial/VP1 and pC1300-CTB-O/VP1 were constructed.(2) Maize embryogenic callus of "416051" and "18-599W" were transformed through genegun-mediated transformation.After screened with increasing hygromycin concentration for three times,164 and 148 resistant calli of"416051" and "18-599W" were obtained.The transformation rate of callus was 23.5%and 11.2%.8 and 25 seedlings were regenerated respectively from resistant calli cultivated in the differentiation media. However,no seedling showed positive when detected by PCR.Maize embryogenic callus of "416051" and "18-599W" were transformed through agrobacterium-mediated transformation.After screened with increasing hygromycin concentration for three times, 340 and 370 resistant calli of"416051" and "18-599W" were obtained.The transformation rate of callus was 10.2%and 8.2%.egeneration capacity of callus from "18-599W" was significantly higher than that from "416051".17 and 73 seedlings were regenerated respectively from resistant calli cultivated in the differentiation media.However,only one "18-599W"seedling showed positive when detected by PCR.(3) It was found in this study that the resistance calli infected by agrobacterium got the higher rate after subcultured for 7-9 days.Sterilization was relatively easy and callus browning rate was lower when agrobacterium concentration was OD600=0.5 and the infection time was 10 min,which was the more favorate condition for transformation.The optimized time for co-culture was 3 d respecitively.The purpose of screening was achieved when Hyg concentration was 10 mg/L.Plant growth was controlled by Paclobutrazol and ABT,and 2.5-3 cm height plants were moved to the medium for seedling growth.Plant resistance was improved by the regulator,and its survival rate reached 25%.