Characterization And Functional Analysis Of A LEAFY Homologous Gene In Longan (Dimocarpus Longan Lour.)

Longan(Dimocarpus longan Lour.)is a member of the family Sapindaceae,and one of the most important subtropical orchard fruit.LEAFY(LFY)homologous gene is one of genes which control the formation of flower meristem,it plays a major role in the process from vegetative growth to reproductive growth,and LFY gene is also considered to relate with the dominating of flower buds growth.In order to explore the molecular machanism of flower buds differentiation and floral reversion in longan,further studies including longan LFY homologous gene cloning,the funcion research of longan LFY homologous gene in the process of flower buds differentiation and floral reversion were performed,and the main results showed as follows:1.The optimizing of longan genomic DNA isolation.The study consulted the isolation methods of other fruit trees,and improved them with combining the specialty of longan.We adopted modified STE method,modified SDS-KAc method and the KIT method to extract genomic DNA from longan leaves.The result showed that modified SDS-KAc method can effectively remove secondary substances including protein,amylose etc.in longan leaves,and obtain genomic DNA with high qualiy and yield.2.Cloning the 5′terminal sequence and the genome sequence of longan LFY gene.Using longan genomic DNA as template,we adopted genome walking to obtain the 5′terminal sequence, it was 1516bp in length.The sequence had high homology with other plant LFY homologous gene, we confirmed the starting position of longan LFY coding region.3.cDNA cloning of longan LFY gene.On the basis of the 5′terminal sequence and 3′sequence,we designed specific primers and cloned the cDNA full sequence of longan LFY gene(LLFY)by RT-PCR.The sequence had high homology with other plant LFY homologous gene, the full sequence was 1184bp in length,containing a 1167-nucleotide of open reading frame (ORF),encoding a putative protein of 388 amino acid residues.We got the genome sequence by spliced the 5′terminal sequence and the sequence obtained by WANG Qing-lian etc.The genome sequence was 3395bp in length,it contained two introns,one was 536bp in length,another was 1391bp.4.The function research of longan LFY gene.Expression patterns of longan LFY gene in the process of inflorescence differentiation and floral reversion revealed by semi-quantitative RT-PCR analysis,and compared expression patterns of the grafting shoot of "honghezi" and "sijimi".The result showed that the expression of longan LFY gene increased gradually in process of inflorescence differentiation,whereas it decreased in process of floral reversion.In addtion,there was no expression of longan LFY gene in the "honghezi" grafting shoot,while in the "sijimi" grafting shoot it showed a feeble expression,it showed LLFY related to inflorescence differentiation and growth.5.Construction of longan LFY gene plant expression vector.Longan LFY gene was cloned into expression vector pC1301PMI-GUS which GUS gene was digested,then we gained plant expression vector of pC1301PMI-LLFY.