Establishment Of Genetic Transformation System Of Lettuce And The Introducing Of HA, NA Antigen Gene Of Avian Influenza

Avian influenza is one kind of which causes the respiratory system disease primarily by the influenza virus violates,is one of the most important infectious diseases to harm feather health。At present,using the transgenic plant products edible vaccine has become a hot spot in this Field.HA and NA are Avian influenza virus important protection antigen genes.It stimulate organism to produce the neutralizing antibody and protect the poultry effectively.Thus lettuce as a bioreactor to large-scale production of edible plant vaccine is an important theoretical and practical value.In this study,based on pBI121 plasmid,respectively constructed with NA / HA gene expression vector of pBI121-NA and pBI121-HA.Using Agrobacterium-mediated genetic transformation methods introduced HA,NA antigen Gene of Avian influenza into lettuce. The results are as follows:1.The plant binary expression vector pBI121 -NA and pBI121 -HA were constructed harboring the Avian influenza antigen gene NA/HA and plasmid pBI121,driven by CaMV 35S promoter.2.Lettuce cotyledon as explants,plant expression vector were used for Agrobacter -ium tumefaciens-mediated transformation Through to changing and exploring extemal environment in the transformation process like Agrobacterium density,the dipping time, co-culture time,pressure choice of antibiotics and so on,we determined and established the highly effective transformation system:the 3 days cotyledons were dipped ten minutes in concentration OD₆₀₀ value of 0.4～0.6,and co-cultured 2～3 days,then cultured on induction screening which includes the 100mg/L cefezolin sodium and 110mg/L Kanamycin.3.At present,a total of 105 resistance kanamycin plants have been obtained, including 6 transgenic plants examined by PCR.GUS dyeing transgenic plants, transgenic plants of leaves and stems are dyed.By RT-PCR detection methods,transgenic plants further verification.The foreign genes have not only integrated into the genome of lettuce,but also to normal expression.