Transformation Of Wheat With Structural Protein Genes From Avian Influenza Virus (H5N1)

In recent years, frequent outbreaks of avian flu around the world caused huge losses, the main characteristics H5N1 of the highly pathogenic avian influenza (HPAI) are not only infect birds, but also mammals and human.Therefore,the development of efficient and inexpensive for AI vaccines has great significance.With the development of modern genetic engineering technology, Plants as bioreactors producing vaccines become research focus, compared with traditional vaccines, it has the advantages of low cost, higher security, convenient to use, direct oral.In this article, the seed-specific expression vector pBar-LYA-AsGlo, pPMI-LYA-AsGlo, p-LYA-AsGlo are constructed for the preparation of the plasmid containing selection marker gene PMI or BAR, and the minimal express cassettes containing target gene. The wheat Z9023, Y12, X48 are used as receptor material, Genes of hemagglutinin (HA), neuraminidase (NA), matrix proteins (M1,M2) of avian influenza H5N1 and selectable genes were transformed into wheat immature embryos by particle bombardment. Then the callus were transferred to selection MS media containing mannose or PPT respectively, by multiply PCR and Southern-blot analysis, The results supported that the exogenous genes were integrated into the wheat genome. Main results are the followings:1. Total 389 resistant lines were obtained:Z9023 have 265 lines; Y12 have 96 ones; X48 have 28 ones.By multiply PCR analysis,25 resistant lines were positive, transformation efficiency was 0.62%、0.43%、0.24%. PCR analysis demonstrated the integration of exogenous genes preliminarily.2. Comparison of mannose/PMI and the PPT/Bar screening system:The results showed:transformed callus in the mannose/PMI system have the capacity of stronger differentiation regeneration; resistant plants were more robust, have more developed root system.3. By extracting the T1 transgenic plants DNA, it was used for southen-blot. The results of southern-blot showed:hybridization membrane containes target gene, southern-blot analysis further demonstrated the integration of exogenous genes.