Cloning And Expression Analysis Of Several Important Genes Involved In Grapevine Floral Development

Grape is among important fruit trees in the world and is also one of the most important deciduous fruit trees in China. The vine is strong in adaptability, early bearing and highly beneficial. Since the reform and opening policy started, the Chinese grape industry has developed very rapidly. For a long time, the time of fertilizer application in grapevine, especially by farmers has been unreasonable since fertilization was based on traditional ideas with no scientific basis. Traditional fertilizer application is based on analysizing mineral elements in leaves and/or soil, and supplementary application of the elements when the levels are below the lower limit of defined ranges.The theory and technology of biochemistry and molecular biology have been used in diagnosis, treatment and prevention of diseases, with modern molecular biology techniques providing a possibility for us to define grapevine phenology using gene information at the molecular level. This study combined with research focus of model plants, researched several important genes of grapevine flower development, cloned full-length cDNA of nine genes, analysis sequences, subcellular localization and expression, and then defined grapevine phenology. The fertilizer test has proved that it is feasible to analyse grapevine phenology by using gene information. The main results of the study are as follows:1. Based on the nucleotide sequences of seven important genes (VvAG, VvAP3, VvFLC, VvFT, VvSOCl, VvAPl, VvFUL) involved in grapevine flower development deposited in Genebank, seven pairs of primers were designed and used to amplify their homologues in grapevine cultivar’Xiangyue’. All the PCR products were ligated to pMD18-T vector and then the recombinant vector was transformed into Ecoli DH5a strain. The anticipated clones checked by PCR were cultured and sent for sequencing. The open reading frame sequences of the7genes and the deduced amino acid sequences were aligned with their corresponding homologues deposited in Genebank, and seven phylogenetic trees were constructed. The results indicated that the nucleotide and amino acid sequences of the7genes in different grapevines were different at some loci and they were more identical with the homologues in different plants, suggesting they evolved in different directions.2. The aim of this study was to isolate the open reading frame sequence of VvAG, VvAP3, VvFLC, VvFUL, VvFT, VvAP2and VvSOCl from’Xiangyue’, one of the most popular table grape cultivar, for a preliminary study on their functions. Specific primers RT-PCR method was used to clone genes, and semi-quantitative PCR was used to analyze the expression of genes in different organs and tissues. Recombinant plasmid was introduced into onion epidermal cells by the particle bombardment method with a PDS1000/He. Transformed cells were incubated for24h at25℃in the dark and green fluorescence was monitored under a Laser scanning confocal microscope. The expression results of the genes in different tissues showed that they were expressed ubiquitously in all the organs and tissues, but the expression levels of some were different. VvFT, VvFUL and VvAP3had the highest expression in young fruit, VvAG and VvAP2expression was highest in flower, while expresson of VvSOC1and VvFLC was highest in young leaves. VvSOC1, VvFT, VvFLC and VvAP2combined with GFP were only located in the nuclei of onion epidermal cells, which showed typical characteristics of transcription factors. On the other hand, VvAG, VvFUL and VvAP3combined with GFP were located in both the plasma membrane and nucleus. All these genes were involved in the development both of reproductive and vegetative organs, and showed the nucleus location phenomena by the combination with GFP except VvAG, VvFUL, and VvAP3which exhibited signals in the plasma membrane. These results could provide some important theoretical basis for more peering study on the molecular mechanism of flower development and the function of the genes studied during grapevine flower development.3. Quantitative real-time RT-PCR was employed to analyze the expression of nine important floral genes in grapevine cv.’Fujiminori’, i.e. VvAG, VvAP3, VvFLC, VvFUL, VvFT, VvAP2, VvSOC1, VvAP1and VvLFY. Results show that the summer buds on the shoots cut back could sprout much earlier and initiate flower bud differentiation and development, but the control summer buds could not form floral buds and inflorescence. There were differences between the expression patterns of the nine genes in treatments and control, and the expression of genes in treated bud and shoot samples was basically same as that of their homologous genes in Arabidopsis. This work provides theoretical information for further understanding of the molecular mechanism of flower development in grapevine, for the utilization of the related genes in genetic engineering, and for some positive control on their expression.4. Quantitative real-time RT-PCR was employed to analyze the expression of nine important floral genes in grapevine, with results showing that expression of VvAP1, VvFUL, VvLFY is greatest in the third period, flower drop was decreased significantly after the second fertilizer which also promoted the expression of VvAP3, VvAP1, VvSOC1, VvFT, VvLFY, but inhibited the expression of VvAG and VvFLC. The expression of VvAP3was the greatest in the sixth fertilizer application period while the expression of VvAPl, VvFUL, VvSOCl, VvLFY, VvFLC, VvAG was decreased significantly. Application of fertilizer three times after flowering led to a significant decrease in fruit drop especially at the fifth period, and affected the expression of genes in different degrees. The expression of VvAP3, VvFUL, VvSOCl, VvFT, VvFLC, VvAG decreased significantly in the eighth period, while weight of single fruit and size of fruit were increased after seventh fertilizer. This reveals that fertilizer of key phenology could affect expression of genes in active state at the molecular level and with accurate analysis of gene information; we can define grapevine phenology by using the generated gene information.