Cloning, Structural and Functional Analysis of Vacuolar Invertase Genes (IbVINs) in Sweet Potato (Ipomoea batatas)
HAN Hao-Hao1,*, DUAN Li-Jun1, WEI Yan-Mei1, HAN Dong-Chen1, ZHAO Ren-Na1, DING Yan1, YAN Feng-Ying2
1 Department of Life Sciences, Linfen Vocational and Technical College, Linfen 041000, China; 2 School of Basic Medical Sciences, Changzhi Medical College, Changzhi 046000, China
Abstract：Sweet potato (Ipomoea batatas) is an important food, industrial, and feed crop. The contents and composition of starch and soluble sugars are key determinants of sweet potato quality. Vacuolar invertase (VIN) genes are closely associated with starch-sugar metabolism; however, their functions and regulatory mechanisms in the starch-sugar metabolism of sweet potato storage roots remain to be systematically elucidated. This study conducted a systematic investigation of the vacuolar invertase gene family in sweet potato to elucidate its role in starch-sugar metabolism and carbon partitioning of storage roots. Three IbVIN members were cloned from 7 sweet potato genotypes, their sequences were analyzed bioinformatically, phylogenetic tree was built, and protein secondary/tertiary structures and conserved domains were predicted. Expression patterns were profiled across cultivars, developmental stages, and tissues using qPCR, starch and soluble sugars were quantified by high-performance liquid chromatography (HPLC), and correlation analyses were performed. This study also cloned the 1.5~2.5 kb upstream promoter regions of IbVINs, predicted cis-acting elements, and carried out subcellular localization assays. In Arabidopsis thaliana, Agrobacterium-mediated transgenic lines overexpressing IbVIN1 were generated, and starch and soluble sugar contents were measured in leaves and seeds. The results showed that IbVIN1, IbVIN2, and IbVIN3 all contained the conserved GH32 domain and the catalytic motifs NDPNG, RDP, and WEC, with structural differences among the proteins. The 3 genes were highly expressed in storage roots but weakly expressed in stems, peaking during early-mid root swelling (65~80 d after transplanting), indicating tissue- and stage-specific patterns. At 95 d after transplanting, IbVIN1 and IbVIN2 correlated negatively with starch content and positively with hexoses content, while IbVIN3 correlated positively with hexoses content. At 125 d, all 3 genes showed significant negative correlations with hexoses content, suggesting dynamic regulation of carbon allocation. All 3 promoter types harbored light-, hormone-, and stress-responsive elements, with typeⅡuniquely containing the low-temperature responsive element (LTR) and typeⅢ containing an auxin-responsive element (AuxRE). Subcellular localization confirmed that all 3 proteins resided in the vacuole. Overexpression of IbVIN1 in A. thaliana significantly decreased leaf starch and increased leaf soluble sugars, while promoting the accumulation of both starch and soluble sugars in seeds. Collectively, these findings reveal that the IbVIN family exhibits spatiotemporal expression and stage-dependent roles in carbon partitioning during storage-root development, clarifies its regulatory function in starch-sugar metabolism, and provides a theoretical basis and candidate targets for sweet potato quality improvement and molecular breeding.
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