火龙果<em>HpFBH3</em>结合E-box反式激活<em>HpCO7</em>表达促进成花

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (2): 575-593.DOI: 10.1016/j.jia.2024.07.039

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火龙果HpFBH3结合E-box反式激活HpCO7表达促进成花

收稿日期:2023-06-06 接受日期:2023-08-11 出版日期:2025-02-20 发布日期:2025-01-22

HpFBH3 transactivates HpCO7 via binding to the E-boxes in the promoter and may accelerate flower formation in pitaya

Xiaowei Cai^{1, 2}, Ling Xiao^{1, 2}, Xiangmei Nie^{1, 2}, Qiandong Hou^{1, 2}, Sulin Wen^{1, 2}, Kun Yang^{1, 2#}, Xiaopeng Wen^{1, 2#}

¹Key Laboratory of Plant Resources Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Institute of Agro-Bioengineering/College of Life Sciences, Guizhou University, Guiyang 550025, China
²Guizhou Key Laboratory of Agro-Bioengineering, Guizhou University, Guiyang 550025, China

Received:2023-06-06 Accepted:2023-08-11 Online:2025-02-20 Published:2025-01-22
About author:Xiaowei Cai, E-mail: vera8472@163.com; #Correspondence Kun Yang, E-mail: kyang5@gzu.edu.cn; Xiaopeng Wen, E-mail: xpwensc@hotmail.com
Supported by:
This work was supported by the National Natural Science Foundation of China (32160681 and 32060663), the National Guidance Foundation for Local Science and Technology Development of China (2023-009), the Guizhou Provincial Basic Research Program (Natural Science) (ZK[2022]YB132) and the Foundation of Postgraduate of Guizhou Province, China (YJSKYJJ[2021]057).

摘要/Abstract

摘要：

火龙果是一种藤本仙人掌科植物，因为其对非生物逆境的高耐受及红紫色的果实而广泛种植于全世界。实践中，诱导成花和促进花芽分化往往可以提高作物的产量。然而，相关基因在火龙果中仍有待进一步挖掘。前人研究发现，FLOWERING BHLHs（FBHs）是促进成花的关键转录因子。本工作中，我们在火龙果中共鉴定到8个FBH转录因子。由于近期发生的全基因组加倍事件，火龙果比甜菜和菠菜编码更多的FBH转录因子。系统进化树显示，火龙果FBHs可以被归为3类,其第II类中，石竹目植物编码的FBH转录因子均含有一个不完整的bHLH结构域，来自基因组的散在重复事件，为非典型FBH。Ka/Ks值显示，HpFBHs进化受到纯化选择影响。对HpFBHs的启动子和表达进行分析发现，它们在与花相关的组织中被时空特异地激活，且表达受非生物逆境的影响，说明HpFBHs参与了火龙果的成花过程。因此，我们选择HpFBH1和HpFBH3两个来自TYPE III的典型FBH与来自TYPE II的非典型FBH进行了功能验证。亚细胞定位、酵母双杂与荧光素酶互补实验结果显示，HpFBH3能够在细胞核内与HpFBH1形成异源二聚体。利用生物信息学手段，HpFBHs被预测通过结合启动子上的E-box参与对下游基因的调控，而这类顺式作用元件广泛存在于HpCOs、HpFTs和HpSOC1s的启动子上。RNA-Seq数据显示，这些成花促进因子的表达与HpFBH3高度协同。酵母单杂及双分子荧光素酶报告实验证实，HpFBH3通过结合HpCO7启动子上的E-box元件反式激活其表达。此外，在拟南芥中异位过表达HpFBH3同样促进了成花过程。综上所述，本研究系统分析了火龙果HpFBH转录因子家族,并将其中的典型FBH尤其是HpFBH3鉴定为成花促进因子，有望成为遗传改良火龙果的目标基因。

Abstract:

Hylocereus polyrhizus, also known as pitaya or dragon fruit, is a climbing cactus grown worldwide because of its excellent performance under drought stress and appealing red-purple fruits. In practice, accelerating flower formation and inducing more flowers usually result in higher yield. However, the genes for this purpose have not been well characterized in pitaya. Previously, FLOWERING BHLHs (FBHs) have been identified as positive regulators of flower formation. In the present work, a total of eight FBHs were identified in pitaya. This is a greater number than in beet and spinach, possibly because of the recent whole-genome duplication that occurred in the pitaya genome. The phylogenetic tree indicated that the FBHs could be divided into three groups. In TYPE II, the genes of Caryophyllales encode atypical FBHs and are generated by dispersed duplication. The K_a/K_s ratios indicated that HpFBHs are under purifying selection. Promoter and expression analysis of HpFBHs revealed that they are spatiotemporally activated in flower-related tissues and responsive to multiple abiotic stresses. These results indicated that HpFBHs are involved in the flower formation of pitaya. Therefore, typical HpFBH1/3 from TYPE III and an atypical HpFBH8 from TYPE II were selected for functional verification. HpFBH3 was found to heterodimerize with HpFBH1 in the nucleus using subcellular localization, yeast two-hybrid and luciferase complementation assays. With bioinformatic analysis, all HpFBHs were predicted to transactivate downstream genes via binding to the E-boxes, which were frequently detected in the promoters of HpCOs, HpFTs and HpSOC1s. RNA-Seq datasets showed that these flowering accelerators were expressed in coordination with HpFBH3. Yeast one-hybrid and dual-luciferase reporter assays further verified that HpFBH3 transactivated HpCO7 by selectively binding to the E-boxes in the promoter. Moreover, ectopic overexpression of HpFBH3 accelerated flower formation in Arabidopsis. In summary, this study systematically characterized the typical HpFBHs, especially HpFBH3, as positive regulators of flower formation, which could be target genes for the genetic improvement of pitaya.

Key words: pitaya , FLOWERING BHLH , dimerization , transactivation , flower formation

Xiaowei Cai, Ling Xiao, Xiangmei Nie, Qiandong Hou, Sulin Wen, Kun Yang, Xiaopeng Wen. 火龙果HpFBH3结合E-box反式激活HpCO7表达促进成花[J]. Journal of Integrative Agriculture, 2025, 24(2): 575-593.

Xiaowei Cai, Ling Xiao, Xiangmei Nie, Qiandong Hou, Sulin Wen, Kun Yang, Xiaopeng Wen. HpFBH3 transactivates HpCO7 via binding to the E-boxes in the promoter and may accelerate flower formation in pitaya[J]. Journal of Integrative Agriculture, 2025, 24(2): 575-593.

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