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Vacuolar invertase (VIN), degrading sucrose to produce glucose and fructose irreversibly, is a key enzyme in soluble sugar metabolism, and involved in plant growth and development, yield and quality formation, and stress resistance. To investigate the physiological functions of VIN genes in soluble sugar metabolism of red pitaya (Hylocereus polyrhizus), HpVIN1 gene was cloned from the fruit, and sequence comparison, phylogenetic relationship, gene expression, subcellular localization, yeast growth complementation and sucrose degradation activity were analyzed. Based on RT-PCR (reverse transcription-polymerase chain reaction) amplification, the ORF (open reading frame) of HpVIN1 gene with a length of 1935 bp was isolated, which encoded 644 amino acids. Sequence analysis showed that HpVIN1 contained key domains related to sucrose degradation activity of invertase, as well as vacuole localization sequences in the N terminal. Phylogenetic analysis suggested that HpVIN1 was closely related to VIN genes from kiwifruit, loquat, apple and grape. Real time fluorescence quantitative PCR detection revealed that HpVIN1 was highly expressed in stems and fruits during the veraison stage (20~25 days after flowering), and weakly expressed in ripen fruits (30 days after flowering). The transient expression of HpVIN1 fused by green fluorescent protein in Arabidopsis thaliana mesophyll protoplasts demonstrated that HpVIN1 protein was localized in the tonoplast and vacuole. By over-expressing in the yeast strain with sucrose utilization defects, HpVIN1 could restore the yeast growth using sucrose as the sole carbon source, which proved that HpVIN1 had the sucrose degradation activity. In vitro catalytic experiment using the yeast total protein suggested that HpVIN1 could degrade sucrose into glucose and fructose. The sucrose degradation activity was the highest at pH 4.0, and rapidly decreased as pH value increasing. The results show that HpVIN1 that locates in the vacuole, has the enzyme activity of degrading sucrose to produce glucose and fructose, and participates in sucrose catabolism of stems and fruits during the veraison stage.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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