Cloning And Function Analysis Of LFY, UFO, FUL Genes In Cinnamomum Camphora

Cinnamomum camphora, a common evergreen tree, is the representative tree of Laurales, Lauraceae and Cinnamomum. The mature Cinnamomum camphora has a egg shape, luxuriant leaves and spreading branches. Moreover, it smells sweet. Therefore, Cinnamomum camphora is widely used in the landscaping, as a common garden tree in the middle and lower reaches of Yangtze River in china. However, after having been ripe, the fruits of Cinnamomum camphora drop from the tree and fall on pedestrians, cars and the ground, which brings inconvenience to the daily life and pollution on the ground. As to solving this problem, this research separated and cloned the homologous genes of LFY, UFO and FUL, called CcLFY, CcUFO, CcFUL, which are the key genes for the formation of flowers and fruits, and analyzed their functions. As a result, this research can provide some guiding molecular bio-information for obtaining Cinnamomum camphora with less flowers and fruits, even with no flowers and fruits in the future.In the first, CcLFY, CcUFO, CcFUL was obtained and analyzed through phylogenetic tree. Then, semi-quantitative RT-PCR analysis was performed for the purpose of finding out functions of these three genes. In final, expression vectors including the target sequences were constructed and introduced into Arabidopsis thaliana, and these phenotypes of transgenic Arabidopsis thaliana were observed and analyzed in order to identify functions of those three genes. In addition, this research also carried out an experiment of yeast two hybrid, so as to make sure whether there is an interaction between CcLFY and CcUFO. The main results of this research were as follows:Firstly, these three genes were obtained as follow: CcLFY through common PCR; CcUFO through flank amplification and common PCR; CcFUL through homology-based cloning, flank amplification and common PCR.Then, phylogenetic trees were constructed in the base of acquired sequences, so as to analyze the gene sequences. As a result, it showed that those three genes have a close relationship with dicotyledon.Secondly, the RNA in different parts of Cinnamomum camphora were collected and extracted by the means of CTAB. Then, Semi-quantitative RT-PCR analysis was performed. It found out that those three genes have an influence on the floral development and formation of fruits in Cinnamomum camphora.Thirdly, 35s-CcLFY, 35s-CcUFO, 35s-CcFUL were successfully constructed by means of tranditional DNA recombination. Then, the electric shock was used to transform these recombinant plasmids into the Agro-bacterium Tumefaciens strain GV1303. Finally these recombinant plasmids were transformed into Arabidopsis thaliana through Agro-bacterium Tumefaciens mediated method. As a result, 8 positive transgenetic Arabidopsis thaliana containing CcLFY, 11 positive transgenetic Arabidopsis thaliana containing CcUFO, and 6 positive transgenetic Arabidopsis thaliana containing CcFUL were obtained. These CcLFY and CcUFO transgenetic Arabidopsis thalianas have early flowering and their lateral branches turn into flowers. What’s more, some CcLFY transgenetic Arabidopsis thaliana have fewer branches, and their branches directly develop into flowers in the bottom without elongating. These CcUFO transgenetic Arabidopsis thaliana also appear short and small stature. In addition, there are late flowering, short and curling fruits in the CcFUL transgenetic Arabidopsis thaliana. These abnormal phenotypes in transgenetic Arabidopsis thaliana show that these three genes have an important effect on the floral development and formation of fruits in Cinnamomum camphora.Fourthly, it showed that there was no gene interaction between CcLFY and CcUFO in the yeast two hybrid experiment.