Cloning and functional analysis of the VdERD6L15 gene in Vitis davidii

Abstract: 【Objective 】Vitis davidii Foëx. is a key wild grape germplasm resource in southern China, the sugars in the V. davidii berry are mainly accumulated in the form of glucose and fructose in the vacuole during the ripening period, and this process is mainly regulated by sugar transporter proteins. The Early- Response to Dehydration six- like (ERD6L) is a subfamily of Monosaccharide Transporters (MSTs), which has been proved as a key regulator in soluble sugar homeostasis. In this study, to investigate the role of VdERD6L15 gene in the growth and development of V. davidii berries, the coding sequence (CDS) and promoter of VdERD6L15 gene were cloned from Xiangci No. 1, and its function and promoter activity were further analyzed.【Methods】The Pearson correlation coefficients and FDR values of the expression data of VdERD6LI5 gene and the soluble sugar content were calculated using the R packages GGally and ggplot2. The CDS of the VdERD6LI5 gene was cloned by reverse transcriptionpolymerase chain reaction (RT- PCR), and the sequence was biologically analyzed by ExPasY, TMHMM2.0, and SWISS-MODEL website. After constructing the VdERD6L15 related expression vector, tobacco was transformed to determine the subcellular localization of VdERD6L15, heterologous expression of VdERD6L15 was observed in yeast strain EBY.VW4000 to determine its function, and the core region of VdERD6L15 promoter was determined by GUS staining.【Results】Based on the Pearsoncorrelation analysis of VdERD6L15 gene expression and sugar content, it was found that the expression of VdERD6LI5 was high in the early stage of berry development and low in the later stage, which was significantly and negatively correlated with the soluble sugar accumulation in the berry of V. davidii. The cDNA from the root tissues of V. davidii was used as a template, primers were designed by Oligo7 website, and the PCR amplification was performed. Sequence analysis showed that the length of the CDS of the VdERD6L15 gene was 1461 bp in length, encoding 486 amino acids and belonging to the Monosaccharide Transporter (MST) family. Analysis by ExPASy online website predicted that the protein encoded by VdERD6L15 had a relative molecular weight of 52 614.73, theoretical isoelectric point of 8.34, fat coefficient of 118.91, and average coefficient of hydrophilicity of 0.628, and it was a class of unstable protein. The results of TMHMM online software analysis indicated that VdERD6L15 may contain 12 transmembrane structural domains, and it was hypothesized that VdERD6L15 was a membrane protein. With SOPMA online software to predict the secondary structure of the VdERD6L15 protein, the results showed that the protein was mainly composed of α-helix 49.18%, β-turn 3.10%, extended strand 18.11% and random coil 26.54%. The prediction of the tertiary structure of this protein was carried out by SWISS-MODEL online software. Observation by Confocal laser scanning microscope revealed that the GFP-VdERD6L15 protein was a membrane protein and had a typical vesicular membrane invagination structure, whereas RFP-SYP122 labeled cellular membranes did not have invaginations, indicating that VdERD6L15 was localized to the tonoplast. The heterologous expression of the VdERD6L15 gene in hexose- deficient yeast strain EBY.VW4000 preliminarily found that the yeast strain transformed with pDR196 null load could grow normally on maltose medium, and failed to grow normally on glucose, fructose and sucrose medium. The yeast strain transformed with pDR196- VdERD6L15 grew well on glucose-containing medium, while growth was obviously inhibited on fructose and sucrose medium, confirming that VdERD6L15 was involved in glucose transport. PlantCARE website was used to analyze and predict the cis-acting elements in the promoter of VdERD6L15, the result showed that the promoter contained mainly light response components, drought stress and hormone response components. The results by GUS staining experiments showed that the P1 (-3000 bp), P2 (-2200 bp), P3 (-1500 bp) and P4 (-1000 bp) transformed tobacco leaves in the experimental group could be stained normally, and only tobacco leaves transfected with P5 (-500 bp) could not be stained normally, which further confirmed that the core region on the promoter of the VdERD6L15 was located between -500 bp to -1000 bp.【Conclusion】In this study, the CDS and promoter of VdERD6L15 gene were cloned and its function was analyzed, and it was preliminarily found that VdERD6L15 was a sugar transporter located on the tonoplast and harbored the glucose transportation function. Through promoter deletion analysis of the VdERD6L15, it was found that the core cis- acting element of the VdERD6L15 promoter was located between 500 bp and 1000 bp upstream of the ATG.