中国农业科学 ›› 2025, Vol. 58 ›› Issue (13): 2663-2681.doi: 10.3864/j.issn.0578-1752.2025.13.013

• 园艺 • 上一篇    下一篇

蓝莓VcTPS/VcTPP基因家族鉴定及VcTPS24对花诱导的功能分析

吴新亮(), 吴慧玲, 张绥林, 曹漫, 冯欣, 周冰洁, 侯智霞()   

  1. 北京林业大学林学院/林木资源高效生产全国重点实验室/北京林业大学蓝莓研究与发展中心,北京 100083
  • 收稿日期:2025-02-24 接受日期:2025-04-25 出版日期:2025-07-01 发布日期:2025-07-05
  • 通信作者:
    侯智霞,E-mail:
  • 联系方式: 吴新亮,E-mail:wxl622@bjfu.edu.cn。
  • 基金资助:
    北京市自然科学基金(62522023); 北京林业大学热点跟踪项目(2022BLRD07)

Identification of the VcTPS/VcTPP Gene Family in Blueberries and Functional Analysis of VcTPS24 in Floral Induction

WU XinLiang(), WU HuiLing, ZHANG SuiLin, CAO Man, FENG Xin, ZHOU BingJie, HOU ZhiXia()   

  1. College of Forestry, Beijing Forestry University/State Key Laboratory of Efficient Production of Forest Resources/Blueberry Research and Development Center of Beijing Forestry University, Beijing 100083
  • Received:2025-02-24 Accepted:2025-04-25 Published:2025-07-01 Online:2025-07-05

摘要:

【目的】 海藻糖-6-磷酸合成酶(TPS)是海藻糖(trehalose)合成途径上的关键酶,参与调控萌芽、植株生长、开花和抗逆性等多种生理功能。鉴定蓝莓海藻糖合成途径上编码TPS、TPP的基因,运用分子生物学手段解析VcTPS24调控成花的分子机制,为揭示蓝莓花诱导的糖信号转导途径奠定基础。【方法】 运用生物信息学方法分析蓝莓(Vaccinium corymbosum L.)海藻糖代谢途径中VcTPS/VcTPP的氨基酸长度、基因结构、保守基序、启动子序列及亚细胞定位。通过qRT-PCR分析VcTPS classⅠ类基因在蓝莓花芽分化过程中的表达特性。通过外源施用赤霉素、生长素和4 ℃低温等处理,分析VcTPS24在蓝莓中对激素和低温胁迫的表达响应。通过双萤光素酶试验分析VcSOC1和VcSVP对VcTPS24启动子的调控作用。在烟草中过表达VcTPS24,验证其调控生长和成花的功能。【结果】 从蓝莓中共鉴定出28个VcTPS和19个VcTPP家族成员,均可分为2个亚家族,VcTPS蛋白的氨基酸数量为465—1 382 aa,平均等电点(pI)为6.02。大多数成员具有3个外显子结构;VcTPP家族蛋白的氨基酸数量为241—434 aa,平均等电点为7.48。大多数成员具有11个外显子结构。VcTPS/VcTPP家族成员启动子序列均包括G-Box、P-Box、LTR和circadian等元件,参与光照、激素、胁迫响应及调控植株生长。VcTPS/VcTPP家族成员多定位在叶绿体和液泡中。在蓝莓花诱导过程中,VcTPS classⅠ类基因的表达水平显著上升,其中,VcTPS24在花芽分化全过程中的表达水平均显著高于未分化组。外源IAA、GA3、SA、MeJA和低温胁迫可显著诱导蓝莓苗VcTPS24表达量上调。双萤光素酶试验表明,VcTPS24的启动子活性可被VcSOC1显著提高,而被VcSVP显著抑制。亚细胞定位表明,VcTPS24定位于细胞核和细胞质。在无外源蔗糖环境下,过表达VcTPS24的烟草种子萌发和胚根伸长得到促进,但其幼苗生长会受过量蔗糖阻碍。短日照条件可显著诱导VcTPS24表达,在长、短日照条件下,过表达VcTPS24烟草NtLFYNtFULNtSOC1的相对表达量均有不同程度的增加,其花序的形成时间均显著早于野生型,表明VcTPS24参与成花诱导调控。【结论】 共鉴定出28个蓝莓VcTPS和19个VcTPP家族成员,VcTPS classⅠ基因在蓝莓花诱导期间发挥重要作用。VcTPS24的表达受外源激素影响,且过表达VcTPS24能促进烟草开花,可能与促进成花基因的表达有关。

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