黄瓜CsHXKs基因家族鉴定及CsHKL1.2调控花器官糖积累

doi:10.13560/j.cnki.biotech.bull.1985.2025-1396

摘要/Abstract

摘要：

目的探究黄瓜（Cucumis sativus L.）己糖激酶（CsHXKs）基因家族成员序列特征及表达规律，并验证CsHKL1.2在黄瓜花器官糖积累中的作用，为黄瓜花器官糖代谢调控及品质育种提供理论依据与基因资源。方法对黄瓜CsHXKs进行系统鉴定及生物信息学分析；基于RT-qPCR技术检测不同组织和花器官不同部位及非生物胁迫下CsHXKs的表达情况；利用亚细胞定位和基因沉默（virus-induced gene silencing, VIGS）技术验证候选基因的位置和功能。结果黄瓜CsHXKs家族包含6个成员，分布于4条染色体，编码蛋白长度为371‒547个氨基酸，分子量为40.4‒59.6 kD。系统进化分析将其分为6个亚类，同一亚类成员具有相似的基因结构和保守基序。CsHXKs具有光响应、低温、干旱、激素及防御相关顺式作用元件。在非生物胁迫下，CsHXKs基因呈现显著的诱导表达。CsHXK3和CsHKL3基因在根中高表达，CsHXK1/2、CsHKL1.1/1.2基因在花中特异表达，其中CsHKL1.2在雄花蜜腺中表达量最高。CsHKL1.2定位于叶绿体。沉默CsHKL1.2后，花器官的花药、雄花蜜腺、柱头、雌花蜜腺、子房等部位中的葡萄糖、果糖、蔗糖含量显著升高，其可溶性酸性转化酶、细胞质中性转化酶、磷酸果糖激酶、蔗糖合成酶、蔗糖磷酸合成酶、己糖激酶的酶活性明显下调。结论黄瓜CsHXKs不同成员可能具有不同的生物学功能，其中CsHKL1.2通过调控糖代谢相关酶活性参与花器官糖积累，初步验证了CsHKL1.2在黄瓜花器官糖代谢中的功能。

关键词: 黄瓜, 己糖激酶（HXK）, 花器官, 糖积累, 糖代谢, 生物信息学分析, 组织特异性, 非生物胁迫

Abstract:

Objective To study the sequence characteristics and expression patterns of hexokinase gene family members (CsHXKs) in cucumber (Cucumis sativus L.), and verify the function of CsHKL1.2 in sugar accumulation in cucumber floral organs, so as to provide a theoretical basis and genetic resources for the regulation of sugar metabolism in floral organs and quality breeding of cucumber. Method Genome-wide identification and comprehensive bioinformatics analysis were performed on CsHXKs in cucumber. The expression profiles of CsHXKs in different tissues, various parts of floral organs, and under abiotic stress conditions were detected via reverse transcription quantitative polymerase chain reaction (RT-qPCR). Subcellular localization assay and virus-induced gene silencing (VIGS) technology were used to verify the subcellular location and biological function of the candidate gene. Result The cucumber CsHXKs gene family comprises six members, which are distributed on four chromosomes. The encoded proteins range from 371 to 547 amino acids in length, with molecular weights of 40.4‒59.6 kD. Phylogenetic analysis divided these members into six subgroups, and members within the same subgroup shared similar gene structures and conserved motifs. Cis-acting element analysis showed that the promoters of CsHXKs contain multiple elements related to light response, low temperature, drought, hormone regulation and defense response. Under abiotic stress, CsHXKs genes exhibited significantly induced expression. CsHXK3 and CsHKL3 exhibited high expressions in the roots, while CsHXK1, CsHXK2, CsHKL1.1 and CsHKL1.2 displayed flower-specific expression patterns, among which CsHKL1.2 showed the highest expression in male flower nectaries. CsHKL1.2 was localized in chloroplasts. After VIGS-mediated silencing of CsHKL1.2, the contents of glucose, fructose and sucrose in floral organs including anthers, male flower nectaries, stigmas, female flower nectaries and ovaries significantly increased, while the activities of soluble acid invertase, cytosolic neutral invertase, phosphofructokinase, sucrose synthase, sucrose phosphate synthase and hexokinase in these tissues were significantly downregulated. Conclusion Different members of the cucumber CsHXKs gene family may exert distinct biological functions. Among them, CsHKL1.2 participates in sugar accumulation in floral organs by regulating the activities of sugar metabolism-related enzymes, which preliminarily verifies the function of CsHKL1.2 in sugar metabolism of cucumber floral organs.

Key words: Cucumis sativus L., hexokinase (HXK), floral organ, sugar accumulation, sugar metabolism, bioinformatics analysis, tissue specificity, abiotic stress

桑志鹏, 侯栋, 王慧婷, 岳宏忠, 李亚莉, 颉建明, 路雯, 许照泰. 黄瓜CsHXKs基因家族鉴定及CsHKL1.2调控花器官糖积累[J]. 生物技术通报, 2026, 42(9): 157-168.

SANG Zhi-peng, HOU Dong, WANG Hui-ting, YUE Hong-zhong, LI Ya-li, XIE Jian-ming, LU Wen, XU Zhao-tai. Identification of the Cucumber CsHXKs Gene Family and Regulation of Floral Organ Sugar Accumulation by CsHKL1.2[J]. Biotechnology Bulletin, 2026, 42(9): 157-168.

图/表 9

表1 HXK蛋白的理化性质分析

Table 1 Physicochemical property analysis of HXK proteins

基因IDGene ID	基因名称Gene name	氨基酸数 Amino acid number	分子量 Molecular weight （Da）	等电点pI	不稳定指数 Instability index	脂肪系数 Aliphatic index	总平均亲水性 Grand average of hydropathicity
CsaV3_2G001860.1	CsHXK1	547	59 630.97	6.46	26.92	99.31	-0.007
CsaV3_1G039830.1	CsHXK2	498	54 226.37	6.00	34.78	92.03	-0.070
CsaV3_3G045830.1	CsHXK3	488	52 745.49	5.91	39.63	93.52	-0.007
CsaV3_6G039140.1	CsHKL1.1	502	54 631.98	7.15	41.49	99.76	0.022
CsaV3_1G043010.1	CsHKL1.2	371	40 373.18	5.16	41.80	95.96	-0.012
CsaV3_6G044550.1	CsHKL3	500	54 756.22	5.77	36.65	89.72	-0.142

图1 黄瓜CsHXKs基因家族成员染色体定位（A）、基因结构（B）和保守基序分析（C）

Fig. 1 Chromosomal localization (A), gene structure (B) and conserved motif analysis (C) of the CsHXKs gene family in cucumber

图2 CsHXKs基因家族系统进化树与共线性分析A：水稻（Os）、拟南芥（At）、黄瓜（Cs）、南瓜（Cmo）、甜瓜（Cm）、西瓜（Ci）和西葫芦（Cp）的HXK家族基因的系统进化树分析，红色五角星表示黄瓜CsHXKs家族成员；B：黄瓜基因组内共线性分析；C：黄瓜、拟南芥和水稻之间的共线性分析

Fig. 2 Phylogenetic tree and collinearity analysis of the CsHXKs gene familyA: Phylogenetic analysis of HXK family genes from Oryza sativa (Os), Arabidopsis thaliana (At), Cucumis sativus (Cs), Cucurbita moschata (Cmo), Cucumis melo (Cm), Citrullus lanatus (Ci) and Cucurbita pepo (Cp), red pentagrams indicate members of the CsHXKs gene family in cucumber. B: Collinearity analysis within the cucumber genome. C: Collinearity analysis among cucumber (C. sativus), A. thaliana and Oryza sativa

图3 CsHXKs基因家族成员顺式元件预测

Fig. 3 Prediction of cis-elements in the members of the CsHXKs gene family

图4 黄瓜CsHXKs家族基因在不同组织器官中的表达A：植株不同组织；B：花器官不同部位；热图中的高度代表其在所有组织中的百分比

Fig. 4 Expressions of CsHXKs family genes in different tissues and organs of cucumberA: Different tissues of the plant. B: Different parts of floral organs. In the heatmap, the height indicates the percentage or proportion of each target in all tissues

图5 黄瓜CsHXKs基因家族在非生物胁迫下的表达特性分析A：干旱胁迫；B：盐胁迫；C：低温胁迫；不同小写字母表示差异显著（P<0.05），误差线代表标准差（SD），n=3，下同

Fig. 5 Expression characterization analysis of the CsHXKs gene family in Cucumis sativus under abiotic stressesA: Drought stress. B: Salt stress. C: Low-temperature stress. Different lowercase letters indicate significant differences (P<0.05), error bars indicate the standard deviation (SD), n=3, the same below

图6 CsHKL1.2亚细胞定位A：亚细胞定位；B：叶绿体场局部图片

Fig. 6 Subcellular localization of CsHKL1.2A: Subcellular localization. B: Magnified view of chloroplast

图7 CsHKL1.2的沉默效率以及沉默后对花不同部位糖含量的影响A：沉默效率；B：葡萄糖；C：果糖；D：蔗糖；*P<0.05，**P<0.01，***P<0.001，****P<0.000 1，ns表示差异不显著（P>0.05），下同

Fig. 7 Silencing efficiency of CsHKL1.2 and its effects on the sugar content in different floral tissues after silencingA: Silencing efficiency. B: Glucose. C: Fructose. D: Sucrose; *P<0.05, **P<0.01, ***P<0.001, ****P<0.000 1, ns indicates no significant difference (P>0.05). The same below

图8 沉默CsHKL1.2对黄瓜花不同部位糖代谢相关酶活性的影响A：可溶性酸性转化酶；B：细胞质中性转化酶；C：磷酸果糖激酶；D：蔗糖合成酶（分解方向）；E：蔗糖合成酶（合成方向）；F：蔗糖磷酸合成酶；G：己糖激酶

Fig. 8 Effects of CsHKL1.2 silencing on the activities of sugar metabolism-related enzymes in different floral parts of cucumberA: Soluble acid invertase. B: Cytoplasmic neutral invertase. C: Phosphofructokinase. D: Sucrose synthase (cleavage direction). E: Sucrose synthase (synthesis direction). F: Sucrose phosphate synthase. G: Hexokinase

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