Construction And Genetic Transformation Of Lily Binary Virus Resistant Vector

Virus infection can decrease the yield and quality of lily, resulting in great economic losses. Nowadays, it has become a serious problem in the production of lily. The development of RNA-mediated technologies may provide a strategy to generate viral resistant Lilies.In this work, the LSV gene (439bp) and LMoV gene (376bp) were separately amplied by reverse transcription polymerase chain reaction (RT-PCR) using the total RNA of virus-infected Lily leaves as template. The LSV-LMoV fusion gene fragment, which size is approximate 800bp, was amplied by overlapping PCR technique, This LSV-LMov fusion gene fragment was cloned into pDONR201 vector by BP reaction using Gateway gene cloning technology, and then was subcloned into pH7GWIWG2(â…¡) vector by LR reaction, resulting in pH7GWIWG2(â…¡)-LSV-LMoV. The construction of pH7GWIWG2(â…¡)-LSV-LMoV vector, which is a binary vector contained LSV and LMov virus, was certified by PCR and sequencing analysis.In order to use real-time quantitative PCR (real-time qPCR) to dectect the transformation efficienc of lily, it is necessary to determine the appropriate control gene used in real-time qPCR. We chosed four control genes commonly used in Lily as screeing samples. First, these four control genes were cloned using gene cloning technology, and then, theit expressions were analyzed at the mRNA level by real-time qPCR using roots, bulblets and leaves of 'Sorbonne' and 'White heaven' Lilies as test materials. Through the analysis of Rotor-gene programmer, it was found that the expression of Actin was the highest stability among these four control genes. Therefore, the Actin was chosen as control gene and then, we established the real-time qPCR assay for dectecting the transformation efficienc of lily.We chosed the sterile sub-scales of 'Sorbone' and 'White heaven' lily as receptor of transformation, Agrobacterium tumefaciens EHA105 as transformation strain, hygromycin gene as selection marker gene. LSV-LMoV fusion gene was transformed into Lily by using RNAi plant expression vector pH7GWIWG2 (â…¡)-LSV-LMoV. The transgenic lilies were detected using RT-PCR and real time PCR technology. The results indicated that 17 'Sorbone' and 6 'White heaven' liies were determined to be positive. From this work, we gain the resistant Lilies, however, the ability of resistance would be researched more.