Studies On Construction Of Vectors Which Resist Cucumber Mosaic Virus And Lily Mottle Virus Mediated By RNA Interference And Transformation Of Lily

Lily is an important economic crop with ornamental, edible and officinal value. However, it isextremely vulnerable to be infected with virus such as Cucumber mosaic virus(CMV)and lily mottlevirus(LMoV).Virus infection leads to a serious impediment to the production of ornamental and ediblelily and substantial economic loss to the lily planting. Using RNAi mediated virus resistance, efficientexpression vectors were built and transgenic lily plants resisting CMV and LMoV were obtained. Theseworks laid a foundation for gaining resistance antiviral germplasm, cultivating antiviral lily varietiesand researching the resistant genetic law of transgenic lily plants mediated by RNAi mediated virusresistance.In this work, partial fragments of CMV2b gene and LMoV cp gene were selected as the RNAinterference sections. Ultimately the RNAi plant expression vectors resisting CMV and LMoV withinverted repeat sequences of the target gene fragments were built successfully. And the originalagrobacterium tumeraciens-mediated genetic transformation system for lily was optimized to improvethe transformation efficiency. And the RNAi plant expression vectors were transformed into lily.Finally, lily transgenic plants resisting CMV, LMoV were obtained. The main results are as follows:1. RNAi plant expression vectors anti CMV, LMoV were built successfully.The partial fragments of CMV2b gene(271bp) and LMoV cp gene(428bp) were selected as theRNA interference sections. Different restriction enzyme cutting sites were used to control the directioninsertion of target fragments. Respectively, the positive and reverse target fragments were connectedwith RNAi plant expression vector pFGC5941which contained an intron. Ultimately the resisting CMVand LMoV RNAi plant expression vectors pFGC5941-C2and pFGC5941-L2with inverted repeatsequences of the target gene fragments were built successfully. The RNAi vectors could be transformedinto lily by agrobacterium tumeraciens.2. Genetic transformation system mediated by agrobacterium tumeraciens in the lily Oriental’Sorbonne’ was optimized.The vector pFGC5941contains resisting glufosinate ammonium gene, it was viable screeningpositive strains by glufosinate ammonium3.0mg/L as the resistance screening concentration ofagrobacterium tumeraciens mediated transformation for lily Oriental ’Sorbonne’ was determinedthrough the concentration selection. Sonication-assisted agrobacterium-mediated transformation wasused to obtain transgenetic lily. In this work, the ultrasonic power90w with10s brought the highestefficiency of transformation.3. Gain the resistance plants of lily Oriental ’Sorbonne’The recombinant agrobacterium tumeraciens infection, cultivation, glufosinate ammoniumresistance selection, enrichment culture were carried on lily Oriental ’Sorbonne’ sterile scales. Theresistant lilies respectively transformed with pFGC5941-L2and pFGC5941-C2were gained after thepreliminary screening. 4. The resistant plants of lily were detected by molecular technology, antiviral transgenic plantswere finally obtained.The resistant plants which obtained by glufosinate resistance screening were detected by PCR andRT-PCR. Preliminary evidence that target genes had been integrated into the genome of lilies and targetgenes in the transgenic plants were expressed at transcription level were identified. Finally the2strainstransgenic plants of lily with LMoV cp gene and1strains transgenic plants of lily with CMV2b genewere obtained.