Cloning, Subcellular Localization And Expression Analysis Of Two Important SBP Genes From Grape (V. Vinifera×V. Labrusca)

Grape belonging to the Vitaceae family, is an important fruit in the world. Grape is the fourth flowering plant after Arabidopsis Thaliana, Oryza sativa and Populus trichocarpa to have its whole genome sequenced. The grape genome is small (approximately500Mbp), which is about3or4times bigger than that of Arabidopsis Thaliana. This makes grape a plant of focus in molecular biology research areas.Eukaryotic transcription factors are among proteins involved in gene transcription, with some that respond to stress and are involved in development having been isolated. Examples include the SBP gene family. The SBP gene family comprises important transcriptional factors in plants with many biological functions, such as plant growth and reproductive organ formation. Despite this, the research of grape SBP gene family is just at its infancy. In order to further elucidate the structure and function of this group of genes, we utilized bioinformatics to analyze the grape SBP gene family. Using electronic cloning and3’RACE, we derived two full length cDNAs of Vv-SPL9and Vv-SPLIO from’Summer Black’. Sub-cellular location analysis was done using expression vectors of SPL9and SPL10constructed to detect their nuclear localization function. Semi-quantitative and fluorescent quantitative RT-PCR were then used to determine expression levels of Vv-SPL9, Vv-SPLIO and microRNA156a (Vv-miR156a) in different organs of grapevine and the correlation between the genes and microRNA156a. The major findings are as follows:1. Bioinformatic analysis of the SBP gene family in grape:Based on the conserved region of Arabidopsis Thaliana SBP protein sequences deposited in the NCBI database, we used Pfam software and retrieved45amino acid sequences on grape, about half of which have grape microRNA156a (Vv-miR156a) recognition sites. Positioning, amino acid composition, physical and chemical properties as well as spatial structure analysis were studied and a preliminary understanding of grape SBP gene family derived.2. Using electronic cloning and3’RACE, we found two full length cDNAs of Vv-SPL9and Vv-SPL10which were then deposited in the GenBank database under accession numbers HM018600and HM018601. The lengths of Vv-SPL9and Vv-SPL10are1649bp and1709bp respectively. The amino acid sequences of both genes highly conserved SBP domains, suggesting that we successfully isolated SPL9and SPL10homologous genes of Arabidopsis Thaliana from grape.3. Sub-cellular location analysis using the expression vectors of SPL9and SPL10constructed show that they were both located in nucleus and had nuclear localization functions.4. Semi-quantitative and fluorescent quantitative RT-PCR detected the expression levels of Vv-SPL9, Vv-SPL10and miR156a in different organs of grapevine with results showing that the two genes were expressed ubiquitously in all organs tested, but at different levels. The expression levels were highest in young fruit, low in middle and large size fruit. The expression levels of Vv-SPL9, Vv-SPL10indicate that these genes may be involved in fruit formation and have some trade-off correlation with those of Vv-miRNA156a in various grapevine organs.