The Cloning And Function Analysis Of Fruit Shape Controlling Key Genes In Eggplant

Eggplant(Solanum melongena) is an important economical vegetable in the world, particularly in Asian and African countries. There is a large amount of eggplant germplasms in the world and they have rich fruit shape diversity. Therefore, eggplant is a good species to study fruit shape. In this thesis, we cloned fruit shape controlling genes YABBY family, SmOvate and Sm WOX13 genes based on Arabidopsis(Arabidopsis thaliana) and tomato(Solanum lycopersicum) existing data, then further analyzed their function and the molecular mechanism of fruit shape controlling in eggplant. This study aims to further enrich fruit shape controlling theory and provide the theoretical guidance for eggplant breeding.1) Due to the fewer genomic data, the research in gene family level is very difficult for eggplant. Tomato and eggplant belong to the Solanum genus of the Solanaceae, and they share a very close genetic relationship. Study of tomato YABBY gene family may provide an important basis for the research of YABBY gene family in eggplant. We scanned the whole tomato genome with YABBY domain, and found nine YABBY members. Among 24 genomic sequenced species, the gene numbers of YABBY family were stable except the seedless plants. The phylogenetic trees constructed in Arabidopsis, rice and tomato showed five distinct clades. Comparison of gene expression patterns in two locules small tomato and more than six locules big size tomato suggested that only SlYAB5 b participated in leaf development, other genes regulated fowler and fruit development, including positive(SlD) and negative(SlFAS, SlYAB1 and SlINO) gene functions.2) Five eggplant YABBY genes(SmYAB2, SmYAB3, SmFAS, SmCRC and SmYAB5) were isolated based on the tomato YABBY gene sequences using homologous cloning. Phylogenetic tree revealed that five eggplant YABBY genes belonged to four clades(YAB1/3, YAB2, CRC and YAB5), and the YAB2 clade included SmFAS and SmYAB2 genes. Gene structures comparison demonstrated that counterparts of eggplant and tomato were very similar, and only intron sizes were different. Transcription profiles of five YABBY genes were carried out in leaf, flower and fruit, and it was found that the expression of YABBY family genes was closely related to fruit development. All the five proteins encoded by YABBY genes were localized in cell nucleus, and revealed that they might share similar function as transcription factors. With the un-conserved region as target sequence, virus-induced gene silence(VIGS) was performed in each YABBY gene. The VIGS results showed that SmFAS and SmYAB5 played the most important roles in eggplant fruit shape regulation, and then SmCRC gene, the SmYAB3 and SmYAB2 genes showed limited effect.3) The eggplant ovate gene(designated as SmOvate) were cloned based on the tomato ovate gene sequence, and it was further compared gene coding sequence, intron, promoter region and expression in round and long fruit shape eggplant cultivars. Between the two type of fruit shape cultivars, there was no significant single nucleotide polymorphisms in 1,077 bp coding sequences, 467 bp intron sequences and 1,494 bp immediate upstream of the transcription start site. According to the gene expression between round and long eggplant cultivar, the expression levels had the most significant difference in the flowers at the 5th day in anthesis. The phylogeny of ovate gene sequences including five eggplant relatives and other species revealed that SmOvate gene was an ortholog of Arabidopsis OFP7. Subcellular localization of SmOvate protein that fused with GFP was localized in the nucleus. Yeast two-hybrid showed that SmOvate protein could interact with SmKnox protein, they might form a protein complex as in Arabidopsis to regulate downstream GA20ox1 gene to affect fruit shape development.4) WUSCHEL-related homeobox genes encode the conserved transcript factor family that plays a significant role in plant development. According to the homology-based cloning result, eggplant SmWOX13 gene consisted of three exons and 1233 bp cDNA that included 834 bp coding sequence. Phylogenetic tree showed that SmWOX13 gene was grouped with tomato WOX8 and Arabidopsis WOX13. The fused SmWOX13-GFP protein was localized in cell nucleus indicated that the SmWOX13 protein might function as a transcription factor.This thesis systematically studied the fruit shape controlling genes in eggplant, and found that SmOvate, SmFAS and SmYAB5 were the key genes controlling eggplant fruit shape. Among them, SmOvate related to the length, and SmFAS and SmYAB5 related to girth and length in eggplant. Therefore, this study is very useful and important for the fruit shape breeding in eggplant.