Functional Analysis Of LLAG1 And LLGLO1 And Construction Of Their RNAi Vectors

Lilies are monocots and are among the best known and loved bulbous flowers. Lilies and their relatives make up one of the largest families of flowering plants with important economic value in the cut flower market around the world. All breeders are in favor with shape breeding and long for the new cultivars with higher ornamental value, especially double flowers with its noble and plump beauties. Studies with eudicots of Arabidopsis thaliana and Antirrhinum majus led to the ABC model of flower development. Although the model doesn't suit to monocots, but it has helped to analyze new genes and gene functions in monocots.Although lilies are among the most important ornamental plants, relatively little research has been conducted on lily flower development. Beneditol identified a class C floral homeotic gene LLAG1 from L.longiflorum, and two years later LLGLO1 genes of class B was isolated and characterized by Wu xiaopin. Our research is going to investigate the function of LLAG1 and LLGLO1. We cloned the two genes from cDNA of L.longiflorum, and inserted them into a binary vector pBI121 respectively. After confirmation of the sequence, Tobacco plants were transformed via Agrobacterium tumefaciens. Out of 82 independent kanamycin-resistant of LLGLO1-pBI121 transgenic plants, 40 exhibited phenotypic alterations. These plants were phenotypically similar to wild-type plants during both vegetative and inflorescence development. However, their final size was clearly smaller than wild-type plants. When the flowers were examined, the petals and stamens were shorter obviously than wilds, resulting in the carpel out of the petals and stamens 0.1-0.4cm. When the anthers and stigma were examined, they were both intumescentia, and the similar phenotypes were observed on ovary. But the sepals we expected didn't show any abnormal alternation. All those basically support the facts that LLGLO1 expressed in whorl 1, 2, 3 and mature carpels detected by RT-PCR.RNA interference(RNAi) is a post-transcriptional gene-silencing phenomenon induced by double-stranded RNA that is conserved among various organisms. Using hairpin constructs, dsRNA can be expressed in plants relatively easily, enabling this technology to be widely used as an efficient tool to silence the expression of both endogenous and invading homologous genes. To gain further insight into the two floral homeotic genes LLAG1 and LLGLO1 we have constructed their RNAi vectors respectively. cDNA fragments of 3′-665-1144-5′,3′-662-874-5′from LLAG1 and 3′-450-718-5′,3′-431-803-5′from LLGLO1 were amplified separately as inverted repeats, and then subcloned into the vector pSK+intron with an intron to make up the ihpRNA constructs. The interfering cassette was removed from the vector pSK+intron-genes and cloned into pCAMBIA1301, generating the RNAi vectors, and transformed the RNAi vectors into Agrobacterium tumefaciens for further research.