IQD Family Analysis In Grape(VITIS VINIFERA)and Functional Verification Of VvSUN Gene

Grapes(Vitis Vinifera)is deciduous fruit tree belonging to Vitaceae Vitis.Grape planted widely in China with high nutritional value and loved by consumers.With the increase in consumption levels,some conventional varieties have been unable to meet the needs of consumers,people began to pursue novel and special high-quality fruits.As an important part of appearance quality of fruit,fruit shape got more and more attention which also gave the significant to molecular study of it.SUN gene is the main QTL locus in tomato controling fruit shape.The gene mutant species were oval.SUN gene belongs to the IQD gene family.IQD is a class of calmodulin-binding proteins.IQD proteins have been reported in Arabidopsis,rice,tomato and other plants.IQD gene family has many members and express in a variety of plants,thus we speculate that it has a wide range of functions.In this study,the IQD gene family was comprehensive identified and biological information was analyzed.We selected VvSUN(VvIQD7)to do further study.we cloned VvSUN gene that may be related to fruit shape from 'Tensan' grape.The functional analysis of the gene was studyed by overexpressing in transgenic tomato plants.The major results were showed as following:1.Sixteen putative VvIQDs were identified in the grape genome and unevenly distributed on the ten chromosomes.The VvIQDs encoded hydrophilic proteins,containing 137-634 amino acids,and their isoelectric point ranged from 9.63 to 10.88.In addition,all the VvIQDs contained the IQ67 domain and were predicted to be located mainly in the nucleus.Phylogenetic analysis suggested that these genes could be divided into four groups(I-IV).The same subfamily member contains a relatively cnserved exon/intron distribution and motif composition.2.VvIQD7 and VvIQD8 were found to be highly homologous to SUN in tomato through sequence alignment.In this study,we searched the grape sequence in NCBI,found the highest similarity sequence of SUN gene in grape as compared with tomato and selected VvIQD7 to designed the specific primer.The cDNA of VvIQD7 gene,which is named VvSUN,was cloned by RT-PCR technique.Sequence analysis showed that the VvSUN gene contains a 1278 bp open reading frame(ORF),which encodes a polypeptide of 452 amino acids with a calculated molecular weight of 48.11 kDa and an isoelectric point of 10.63.Moreover,the data also indicated that VvSUN is a non-transmembrane and hydrophilic protein and most likely locate in the nucleus and mitochondrial membrane,without signal peptide.Secondary structure analysis indicated that a-helix and random coil are the main structural elements in VvSUN protein.The amino acid sequence encoded by VvSUN gene has an IQ conserved domain,and the sequence similarity to other seven plants IQD family members in NCBI is 43%-53%.The phylogenetic tree showed that grape has the closest relationship with Arabidopsis thaliana IQD12 gene.According to these observations,we predicted that VvSUN protein in grape has similar functions with SUN protein in tomato.In addition,we performed quantitative real-time PCR(qRT-PCR)to determine the expression pattern of VvSUN in grape at different developmental stages and under gibberellin(GA3)treatments and used the vertical and horizontal diameters to evaluate the effects of GA3.Real-time PCR analysis revealed that the expression level of VvSUN gene showed the highest level at anthesis in two varieties,while maintained relatively low levels after anthesis.GA3 treatment markedly increased the fruit shape index,and pronouncedly enhanced the expression level of VvSUN gene in young fruit.Thus,our results suggested that the VvSUN gene in grape involve in the regulation of fruit elongation.3.The plant expression vector pYH4215-VvSUN was successfully constructed with the recombinant DNA technology,and the vector was transducted into the agrobacterium stain EHA105.Tomato transgenic plants were obtained by the Agrobacterium-mediated method.GUS staining detected five positive plants.Following the PCR and RT-PCR detection,two transgenic positive plants were obtained at last.The results showed that the two positive transgenic plants containing the VvSUN gene,indicating that pYH4215-VvSUN plasmid T-DNA region successfully integrated into the tomato genome.Expression analysis of transgenic tomatoes showed that the expression of VvSUN began to rise at the stage of anthesis,and had high expression in young fruit period,while the expression was lower before anthesis and middle-late stage of fruit development.The observe of phenotype showed that changes occurred in floral organs.Flowers and fruits became slender.These results suggested that VvSUN gene control the elongated fruit shape.