关键词: 蓝莓（Vaccinium corymbosum L.）, 海藻糖, 双萤光素酶试验, 海藻糖-6-磷酸合成酶, 花诱导

Abstract:

【Objective】 Trehalose-6-phosphate synthase (TPS), as a key enzyme in the trehalose synthesis pathway, is involved in regulating various developmental process such as germination, plant growth, flowering, and stress resistance. In this study, genes encoding TPS and TPP in the blueberry trehalose synthesis pathway were identified. We analyzed the molecular mechanism of VcTPS24 in regulating floral induction through molecular biology methods, laying the foundation for revealing the sugar signaling pathway regulating blueberry floral induction. 【Method】 Bioinformatics methods was used to analyze the number of amino acid, gene structure, conserved motifs, promoter sequences, and subcellular localization of VcTPS/VcTPP family members in the trehalose metabolism pathway of blueberries (Vaccinium corymbosum L.). The expression characteristics of VcTPS class I genes during blueberry floral differentiation were analyzed through qRT-PCR experiments. We analyzed the expression of VcTPS24 in blueberries under hormone and low temperature stress by exogenous application of gibberellin, auxin and other hormones, and 4 ℃ low temperature treatment. The regulatory effects of VcSOC1 and VcSVP on the VcTPS24 promoter were analyzed by dual luciferase assay. We validated the function of VcTPS24 in regulating growth and floral induction by overexpressing it in tobacco. 【Result】 A total of 28 VcTPS and 19 VcTPP family members were identified from blueberry, which can be divided into two subfamilies. VcTPS family members encoding proteins ranging from 465 to 1 382 amino acids, with an average isoelectric point (pI) of 6.02. Most members have 3 exon structures. VcTPP family members encoding proteins ranging from 241 to 434 amino acids, with an average isoelectric point (pI) of 7.48. Most members have 11 exon structures. Cis acting elements such as G-Box, P-Box, LTR, and circadian, which are involved in light, hormone, stress response, and regulation of plant growth, were found in the promoter sequences of VcTPS/VcTPP family members. Members of the VcTPS/VcTPP family are mostly located in chloroplasts and vacuoles. The expression level of VcTPS class I genes significantly increased during blueberry floral induction, and the expression level of VcTPS24 was significantly higher through the entire period of flower bud differentiation than in the undifferentiated group. Subcellular localization assay showed that the expression of VcTPS24 can be significantly upregulated in blueberry seedlings by exogenous IAA, GA₃, SA, MeJA, and low temperature stress. The dual luciferase assay showed that the promoter activity of VcTPS24 was significantly increased by VcSOC1, while it was significantly decreased by VcSVP. VcTPS24 is located in the nucleus and cytoplasm. Under the condition of no exogenous sucrose supply, the germination and radicle elongation of tobacco seeds overexpressing VcTPS24 were promoted, but excessive sucrose hindered the growth of tobacco seedlings overexpressing VcTPS24. The expression level of VcTPS24 was upregulated under short day condition. Under both long and short day conditions, the relative expression levels of NtLFY, NtFUL, and NtSOC1 in overexpressing VcTPS24 tobacco lines were all increased to varying degrees, and the formation time of their inflorescences was significantly earlier than that of the wild type. This indicates that VcTPS24 is involved in the regulation of floral induction. 【Conclusion】 A total of 28 blueberry VcTPS and 19 VcTPP family members were identified, and the VcTPS class I gene played an important role during blueberry floral induction. The expression of VcTPS24 was influenced by exogenous hormones, and the overexpression of VcTPS24 can promote tobacco flowering, which may be related to the upregulated expression of flowering genes.

Key words: blueberry (Vaccinium corymbosum L.), trehalose, dual luciferase reporter assay, trehalose-6-phosphate synthase, floral induction