Identification of the Cucumber CsHXKs Gene Family and Regulation of Floral Organ Sugar Accumulation by CsHKL1.2

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

Abstract

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. Subcellular localization results demonstrated that CsHKL1.2 is 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

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

Figures/Tables 9

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

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

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

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

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

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

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

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

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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