无患子SmHD-ZIP25和SmHD-ZIP29基因在花发育中的功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2026-0174

摘要/Abstract

摘要：

目的探究无患子（Sapindus mukorossi）SmHD-ZIP25和SmHD-ZIP29在花发育及性别分化中的调控作用，为无患子分子设计育种提供有价值的参考基因。方法以无患子为材料，克隆HD-ZIP I亚家族成员SmHD-ZIP25和SmHD-ZIP29，并对其进行序列比对、系统发育、亚细胞定位、表达模式分析，并通过拟南芥异源转化探究这2个基因功能。结果序列比对和系统进化树分析显示，SmHD-ZIP25和SmHD-ZIP29具有完整的Homeodomain和Leucine Zipper结构域，且与参与性别分化的油柿MeGI基因高度同源；亚细胞定位表明二者蛋白均定位于细胞核。表达模式分析发现，SmHD-ZIP25和SmHD-ZIP29在无患子雌雄花发育过程中呈现不同的表达规律，在无患子根、茎、叶组织中表达水平相似。与野生型拟南芥相比，过表达SmHD-ZIP25或SmHD-ZIP29均能导致拟南芥出现早花、莲座叶卷曲、果荚发育不良等表型，并显著影响花器官发育。具体表现为雌蕊相对伸长，而雄蕊发育受到抑制（花药短小、花粉败育且活力严重下降）；细胞学观察进一步证实转基因植株花药药室发育异常，花粉粒形态塌陷。RT-qPCR检测表明，这2个基因通过上调开花整合因子AtFT及心皮发育基因AtSHP2、AtCRC的表达，同时抑制茉莉酸信号通路关键基因AtCOL1及其下游雄蕊发育核心因子AtAMS和AtMYB21的表达，从而协同调控促雌抑雄的发育进程。结论 SmHD-ZIP25和SmHD-ZIP29是参与调控花器官发育平衡的关键因子，为阐明无患子花发育及性别分化的分子机制提供了新的理论依据。

关键词: 无患子, HD-ZIP转录因子, 花发育, 性别分化, 雌蕊发育, 雄蕊发育, 花粉育性, 分子调控机制

Abstract:

Objective To investigate the regulatory roles of SmHD-ZIP25 and SmHD-ZIP29 in Sapindus mukorossi flower development and sexual differentiation, and to provide valuable reference genes for its molecular design breeding. Method Using S. mukorossi as a model plant, two members of the HD-ZIP I subfamily, SmHD-ZIP25 and SmHD-ZIP29, were cloned. These genes were subjected to sequence alignment, phylogenetic analysis, subcellular localization, and expression pattern analysis. Their functions were further investigated through heterologous transformation in Arabidopsis. Result Sequence alignment and phylogenetic tree analysis revealed that SmHD-ZIP25 and SmHD-ZIP29 possessed complete Homeodomain and Leucine Zipper domains and exhibited high homology with the sex differentiation-related persimmon MeGI gene. Subcellular localization studies indicated that both proteins were localized in the nucleus. Expression pattern analysis revealed distinct expression patterns of SmHD-ZIP25 and SmHD-ZIP29 during S. mukorossi male and female flower development, while they showed similar expression levels in root, stem, and leaf tissues. Compared with wild-type Arabidopsis, overexpression of either SmHD-ZIP25 or SmHD-ZIP29 exhibited phenotypes including premature flowering, rosette leaf curling, and pod developmental abnormalities, significantly impairing floral organ development. Specifically, pistils exhibited relative elongation while stamen development was suppressed (shortened anthers, pollen abortion, and severely reduced pollen viability). Cytological observations further confirmed abnormal anther sac development and collapsed pollen grain morphology in transgenic plants. RT-qPCR analysis indicated that these two genes synergistically regulated female-promoting and male-suppressing developmental processes by upregulating the expression of the flowering integrator AtFT and of the carpel developmental genes AtSHP2 and AtCRC, while simultaneously suppressing the expression of the key jasmonic acid signaling pathway gene AtCOL1 and its downstream male organ developmental core factors AtAMS and AtMYB21. Conclusion SmHD-ZIP25 and SmHD-ZIP29 are key factors involved in regulating floral organ developmental balance, providing new theoretical support for elucidating the molecular mechanisms underlying S. mukorossi flower development and sexual differentiation.

Key words: Sapindus mukorossi, HD-ZIP transcription factor, flower development, sex differentiation, pistil development, stamen development, pollen fertility, molecular regulatory mechanism

陈宁一, 吴文彤, 赵国春, 贾黎明, 陈仲. 无患子SmHD-ZIP25和SmHD-ZIP29基因在花发育中的功能研究[J]. 生物技术通报, 2026, 42(9): 210-220.

CHEN Ning-yi, WU Wen-tong, ZHAO Guo-chun, JIA Li-ming, CHEN Zhong. Functional Study of SmHD-ZIP25 and SmHD-ZIP29 Genes in Flower Development of Sapindus mukorossi[J]. Biotechnology Bulletin, 2026, 42(9): 210-220.

图/表 10

图1 SmHD-ZIP25、SmHD-ZIP29与柿属同源蛋白的序列比对（A）及系统发育分析（B）图A中红线和蓝线分别表示同源异型结构域（HD）和亮氨酸拉链（LZ）结构域

Fig. 1 Sequence alignment (A) and phylogenetic analysis (B) of SmHD-ZIP25, SmHD-ZIP29 and their homologs from Diospyros speciesIn Fig. A, the red and blue lines indicate the conserved Homeodomain (HD) and Leucine Zipper (LZ) domains, respectively

图2 SmHD-ZIP25与SmHD-ZIP29的亚细胞定位

Fig. 2 Subcellular localization of SmHD-ZIP25 and SmHD-ZIP29

图3 SmHD-ZIP25和SmHD-ZIP29在无患子不同器官中的表达量FF1-FF7指雌花发育的7个时期，MF1-MF7指雄花发育的7个时期。* P < 0.05，** P < 0.01，*** P < 0.001，下同

Fig. 3 Expression of SmHD-ZIP25 and SmHD-ZIP29 in different organs of S. mukorossiFF1-FF7 represent seven developmental stages of female flowers, and MF1-MF7 represent seven developmental stages of male flowers. * P < 0.05, ** P < 0.01, *** P < 0.001, the same below

图4 SmHD-ZIP25过表达拟南芥植株的分子鉴定及表型分析A：转基因植株的PCR鉴定；B：SmHD-ZIP25在野生型与转基因植株中的相对表达水平；C：开花期野生型与过表达植株的整体表型对比；D：20 d龄幼苗的营养生长形态；E：莲座叶形态；F：莲座叶数量与开花时间的统计分析；G：果荚形态（整体和解剖）；H：茎生叶的卷曲表型；比例尺：1 cm。图5同

Fig. 4 Molecular characterization and phenotypic analysis of Arabidopsis plants overexpressing SmHD-ZIP25A: PCR identification of transgenic plants. B: Relative expression of SmHD-ZIP25 in wild-type and transgenic plants. C: Overall phenotype comparison between wild-type and overexpressing plants at the flowering stage. D: Vegetative growth morphology of 20-day-old seedlings. E: Morphology of rosette leaves. F: Statistical analysis of rosette leaf number and flowering time. G: Silique morphology (intact and dissected). H: Curling phenotype of cauline leaves. Scale bar: 1 cm. The same for Fig. 5

图5 SmHD-ZIP29过表达拟南芥植株的分子鉴定及表型分析

Fig. 5 Molecular characterization and phenotypic analysis of Arabidopsis plants overexpressing SmHD-ZIP29

图6 SmHD-ZIP25过表达导致雄蕊发育抑制与花器官形态变化A：花整体及雌、雄蕊表型对比；B：柱头花粉附着情况；C：雌蕊长度与雄蕊数量的统计分析；比例尺：250 µm（A），50 µm（B）。图7同

Fig. 6 Overexpression of SmHD-ZIP25 leads to stamen development inhibition and floral organ morphological changesA: Comparative phenotypes of the whole flower, pistil, and stamens. B: Pollen load on the stigma. C: Statistical analysis of pistil length and stamen number. Scale bars: 250 µm (A), 50 µm (B). The same for Fig. 7

图7 SmHD-ZIP29过表达导致雄蕊发育抑制与花器官形态变化

Fig. 7 Overexpression of SmHD-ZIP29 leads to stamen development inhibition and floral organ morphological changes

图8 SmHD-ZIP25和SmHD-ZIP29过表达植株花药横切面石蜡切片Stage 9：单核小孢子液泡化期；Stage 10：单核小孢子晚期；Stage 11：绒毡层降解起始期；Stage 12：花粉成熟期；Stage 13：花药开裂期。E：表皮；En：内层；ML：中层；T：绒毡层；Msp：小孢子。比例尺：50 μm

Fig. 8 Paraffin sections of anther cross-sections from SmHD-ZIP25 and SmHD-ZIP29 overexpressing plantsStage 9: Uninucleate microspore vacuolation stage. Stage 10: Late uninucleate microspore stage. Stage 11: Tapetum degradation initiation stage. Stage 12: Pollen maturation stage. Stage 13: Anther dehiscence stage. E: Epidermis. En: Endothecium. ML: Middle layer. T: Tapetum. Msp: Microspore. Scale bars: 50 μm

图9 SmHD-ZIP25和SmHD-ZIP29过表达植株花药与花粉的显微结构及育性分析A：花药扫描电镜图；B：花粉粒表面形态扫描电镜图；C：花药亚历山大染色；D：花粉体外萌发情况。比例尺：300 μm（A），10 μm（B），50 μm（C），100 μm（D）

Fig. 9 Microstructural and fertility analysis of anthers and pollen in SmHD-ZIP25 and SmHD-ZIP29 overexpressing plantsA: Scanning electron micrograph of anthers. B: Scanning electron micrograph of pollen grain surface morphology. C: Alexander staining of anthers. D: Pollen germination in vitro. Scale bars: 300 μm (A), 10 μm (B), 50 μm (C), 100 μm (D)

图10 SmHD-ZIP25（A）和SmHD-ZIP29（B）转基因株系花发育内源基因表达变化

Fig. 10 Changes in endogenous gene expression during flower development in SmHD-ZIP25 (A) and SmHD-ZIP29 (B) transgenic lines

参考文献 30

[1]	Yao L, Lu J, Wang J, et al. Advances in biosynthesis of triterpenoid saponins in medicinal plants [J]. Chin J Nat Med, 2020, 18(6): 417-424.
[2]	Liu JM, Zhao GC, Zheng YL, et al. Genetic diversity and adaptive evolutionary history of Sapindus in China: insights from whole-genome resequencing of 100 representative individuals [J]. Plant Biotechnol J, 2025, 23(7): 2485-2500.
[3]	Li JR, Wang HX, Wang LX, et al. Comprehensive analysis of the complete chloroplast genome of the cultivated soapberry and phylogenetic relationships of Sapindaceae [J]. Ind Crops Prod, 2025, 228: 120952.
[4]	Wang X, Zhang RG, Yun QZ, et al. Comprehensive analysis of complete mitochondrial genome of Sapindus mukorossi Gaertn.: an important industrial oil tree species in China [J]. Ind Crops Prod, 2021, 174: 114210.
[5]	Zhao GC, Xu YY, Gao YH, et al. Morphological dynamics of flower development and sex differentiation in monoecious soapberry (Sapindus mukorossi Gaertn.) [J]. Ind Crops Prod, 2025, 235: 121724.
[6]	吴翠翠, 肖水平. 陆地棉HD-Zip家族全基因组鉴定及响应非生物胁迫的表达分析 [J]. 生物技术通报, 2024, 40(2): 130-145.
	Wu CC, Xiao SP. Genome-wide identification of HD-zip gene family in Gossypium hirsutum L. and expression analysis in response to abiotic stress [J]. Biotechnol Bull, 2024, 40(2): 130-145.
[7]	黄丹, 彭兵阳, 张盼盼, 等. 油茶HD-Zip基因家族鉴定及其在非生物胁迫下的表达分析 [J]. 生物技术通报, 2025, 41(6): 191-207.
	Huang D, Peng BY, Zhang PP, et al. Identification of HD-zip gene family in Camellia oleifera and analysis of its expression under abiotic stress [J]. Biotechnol Bull, 2025, 41(6): 191-207.
[8]	Acosta IF, Przybyl M. Jasmonate signaling during Arabidopsis stamen maturation [J]. Plant Cell Physiol, 2019, 60(12): 2648-2659.
[9]	Moore MJ, Soltis PS, Bell CD, et al. Phylogenetic analysis of 83 plastid genes further resolves the early diversification of eudicots [J]. Proc Natl Acad Sci U S A, 2010, 107(10): 4623-4628.
[10]	Akagi T, Henry IM, Tao R, et al. A Y-chromosome-encoded small RNA acts as a sex determinant in persimmons [J]. Science, 2014, 346(6209): 646-650.
[11]	Akagi T, Henry IM, Kawai T, et al. Epigenetic regulation of the sex determination gene MeGI in polyploid persimmon [J]. Plant Cell, 2016, 28(12): 2905-2915.
[12]	Akagi T, Shirasawa K, Nagasaki H, et al. Correction: The persimmon genome reveals clues to the evolution of a lineage-specific sex determination system in plants [J]. PLoS Genet, 2020, 16(5): e1008845.
[13]	Rashid D, Devani RS, Rodriguez-Granados NY, et al. Ethylene produced in carpel primordia controls CmHB40 expression to inhibit stamen development [J]. Nat Plants, 2023, 9(10): 1675-1687.
[14]	Zhao GC, Gao YH, Gao SL, et al. The phenological growth stages of Sapindus mukorossi according to BBCH scale [J]. Forests, 2019, 10(6): 462.
[15]	Piepers M, Erbstein K, Reyes-Hernandez J, et al. GreenGate 2.0: Backwards compatible addons for assembly of complex transcriptional units and their stacking with GreenGate [J]. PLoS One, 2023, 18(9): e0290097.
[16]	Yang HW, Akagi T, Kawakatsu T, et al. Gene networks orchestrated by MeGI: a single-factor mechanism underlying sex determination in persimmon [J]. Plant J, 2019, 98(1): 97-111.
[17]	高亚娇, 赵国春, 贾黎明, 等. 无患子SmCYCD3;2基因的克隆与功能研究 [J]. 北京林业大学学报, 2025, 47(12): 87-96.
	Gao YJ, Zhao GC, Jia LM, et al. Cloning and functional studies on SmCYCD3;2 gene in Sapindus mukorossi [J]. J Beijing For Univ, 2025, 47(12): 87-96.
[18]	Xu YY, Zhao GC, Ji XQ, et al. Metabolome and transcriptome analysis reveals the transcriptional regulatory mechanism of triterpenoid saponin biosynthesis in soapberry (Sapindus mukorossi Gaertn.) [J]. J Agric Food Chem, 2022, 70(23): 7095-7109.
[19]	Czechowski T, Stitt M, Altmann T, et al. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis [J]. Plant Physiol, 2005, 139(1): 5-17.
[20]	Sanders PM, Bui AQ, Weterings K, et al. Anther developmental defects in Arabidopsis thaliana male-sterile mutants [J]. Sex Plant Reprod, 1999, 11(6): 297-322.
[21]	Lian H, Li XR, Liu ZY, et al. HYL1 is required for establishment of stamen architecture with four microsporangia in Arabidopsis [J]. J Exp Bot, 2013, 64(11): 3397-3410.
[22]	Kardailsky I, Shukla VK, Ahn JH, et al. Activation tagging of the floral inducer FT [J]. Science, 1999, 286(5446): 1962-1965.
[23]	Zhu J, Chen H, Li H, et al. Defective in Tapetal Development and Function 1 is essential for anther development and tapetal function for microspore maturation in Arabidopsis [J]. Plant J, 2008, 55(2): 266-277.
[24]	Pinyopich A, Ditta GS, Savidge B, et al. Assessing the redundancy of MADS-box genes during carpel and ovule development [J]. Nature, 2003, 424(6944): 85-88.
[25]	Song SS, Qi TC, Huang H, et al. The jasmonate-ZIM domain proteins interact with the R2R3-MYB transcription factors MYB21 and MYB24 to affect jasmonate-regulated stamen development in Arabidopsis [J]. Plant Cell, 2011, 23(3): 1000-1013.
[26]	Han KY, Lai M, Zhao TY, et al. Plant YABBY transcription factors: a review of gene expression, biological functions, and prospects [J]. Crit Rev Biotechnol, 2025, 45(1): 214-235.
[27]	Mai YN, Sun P, Suo YJ, et al. Regulatory mechanism of MeGI on sexuality in Diospyros oleifera [J]. Front Plant Sci, 2023, 14: 1046235.
[28]	Carabelli M, Turchi L, Morelli G, et al. Coordination of biradial-to-radial symmetry and tissue polarity by HD-ZIP II proteins [J]. Nat Commun, 2021, 12: 4321.
[29]	Zhang T, You J, Zhang Y, et al. LF1 regulates the lateral organs polarity development in rice [J]. New Phytol, 2021, 231(3): 1265-1277.
[30]	Wu ML, Bian XX, Huang BB, et al. HD-Zip proteins modify floral structures for self-pollination in tomato [J]. Science, 2024, 384(6691): 124-130.