Established High-efficient Genetic Transformation System And Studied Of The Stable Inheritance In Transgenic Carrots

Rotavirus　(RV)　infection　is　a　major　cause　of　dehydrating　diarrhea　in　children　all　over　the　world,　especially　in　developing　country.　VP7　and　VP4　are　major　outer　capsid　protein　and　they　are　　primary　candidates　for　inclusion　in　a　subunit　or　recombinant.　Vp6　is　main　virus　protein,　can　induce　body　to　produce　antibody.　So　introducing　VP7,VP4　and　VP6　genes　into　carrot　to　develop　edible　plant　vaccine　of　rotaviruses　would　change　the　traditional　means　of　production　of　vaccines　and　the　cost　of　vaccine　production　would　be　reduced　greatly.　It　is　provided　with　commercial　meaning　and　applicative　prospect.　In　this　study　,　hypocotyls　were　used　as　the　explant,vp4,vp6　and　vp7　genes　were　introduced　into　carrot　by　Agrobacterium　tumefaciens　LBA4404,　and　high-efficient　genetic　transformation　system　of　carrot　were　established,such　as:　Infected　times,　co-cultural　condition,　Kan　sensitive　experiments,　Cab　and　Cef　contrastive　experiments.　Thee　results　indicated　that:　1.　7d　seedling,　2　d　of　co-cultivation　and　15　min　of　infection　of　system.　Media　of　resistant　callus:　B5　+　0.1　mg/L　2,　4-D　+　0.2　mg/L　KT　+　75　mg/L　Kan　+　500　mg/L　Cab.　Media　of　resistant　shoots:B5　+　100　mg/L　Kan　+　500　mg/L　Cab.　Media　of　resistant　root:　B5　+　0.1　mg/L　IBA　+　75　mg/L　Kan　+　300　mg/L　Cef.　2. Hundreds　of　regenerated　Kan-resistant　carrot　plantlets　were　gained　and　analyzed　by　PCR,58　of　156　plantlets　were　confirmed　that　the　VP6　gene　had　been　inserted　into　plant　genome;84　of　172　plantlets　were　confirmed　that　the　VP7　gene　had　been　inserted　into　plant　genome;31　of　80　plantlets　were　confirmed　that　the　VP4　gene　had　been　inserted　into　plant　genome.　3.　Resistant　plants　were　covered　with　plastic　velamen　and　gradually　opened　the　plastic　velamen.　The　resistance　of　plants　grows　well　and　increases.　The　survival　rate　enhanced　35　%.Compare　with　land,　death　rate　in　the　greenhouse　reduced　49.5　%　in　low　temperature　jarovization.　4.　RT-PCR　detection　showed　primarily　the　transcription　of　VP6　and　VP7.T1　plantlets　with　VP6,VP7　genes　were　further　confirmed　by　PCR,PCR-　Southern　and　Southern　blot　that　the　target　gene　had　been　inserted　into　the　carrot　genome.　5. PCR　analysis　for　7　transgenic　plants　in　T1　generation　showed　two　of　them　closed　to　1:1.Other　two　plants　closed　to　1:3.