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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (10): 1628-1638.doi: 10.3724/SP.J.1006.2020.94166

• RESEARCH NOTES • Previous Articles     Next Articles

Promoter cloning and expression analysis of the hexokinase gene CsHXK2 in tea plant (Camellia sinensis)

LI Na-Na1(), LIU Ying1,2, ZHANG Hao-Jie1, WANG Lu1, HAO Xin-Yuan1, ZHANG Wei-Fu1, WANG Yu-Chun1, XIONG Fei1,3, YANG Ya-Jun1,*(), WANG Xin-Chao1,*()   

  1. 1 Tea Research Institute, Chinese Academy of Agricultural Sciences / National Center for Tea Improvement / Key Laboratory of Tea Biology and Resources Utilization, Ministry of Agriculture and Rural Affairs, Hangzhou 310008, Zhejiang, China
    2 College of Horticulture, Northwest A&F University, Yangling 712100, Shaanxi, China
    3 College of Horticulture, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China
  • Received:2019-11-06 Accepted:2020-06-02 Online:2020-10-12 Published:2020-07-03
  • Contact: Ya-Jun YANG,Xin-Chao WANG E-mail:nanali@tricaas.com;yjyang@tricaas.com;xcw75@tricaas.com
  • Supported by:
    National Natural Science Foundation of China(31700615);China Postdoctoral Science Foundation(2017T100119);China Postdoctoral Science Foundation(2016M600150);Earmarked Fund for China Agriculture Research System (Tea)(CARS-19)


Hexokinase is a kind of dual-function protein playing a crucial role in hexose phosphorylation and sugar signaling transduction in plants. In our previous study, four hexokinase genes from tea plant were cloned, and CsHXK2 gene encoded 492 amino acid residues was classified as Type A HXKs together with AtHXK3 from Arabidopsis thaliana and LeHXK4 from tomato. 2029 bp promoter of CsHXK2 gene was obtained by RT-PCR. Further sequence analysis showed that CsHXK2 gene may be regulated by light, low temperature, pathogen, sugars and phytohormones, and specifically expressed in leaves, flowers, seeds, roots, axillary buds. CsHXK2 protein was localized in the chloroplast. Functional complementation of the hexokinase-deficient yeast mutant showed that the CsHXK2 mature protein removal of chloroplast transit peptide, had phosphorylation activity of glucose and fructose. Tissue-specific expression analysis found that CsHXK2 had the highest transcriptional levels in roots and stems, whereas the lowest in old leaves. The expression level of CsHXK2 gene was significantly down-regulated in both cold stress treatment and leaves infected treatment by Colletotrichum camelliae. However, the expression level of CsHXK2 was significantly stimulated after exogenous GA3 treatment in tea leaves. In conclusion, the results suggested that CsHXK2 played an important role in the regulation of both development and stress responses in tea plant.

Key words: tea plant, hexokinase, promoter, subcellular localization, expression regulation

Table 1

Primers information"

Sequence name
Forward primer (5'-3')
Reverse primer (5'-3')
CsHXK2 promoter
Promoter amplification
Subcellular localization
Subcellular localization
Yeast expression
Yeast expression
Colony PCR

Fig. 1

Amplification and sequence of CsHXK2 promoter in the tea plant A: electrophoresis of PCR products of CsHXK2 promoter; B: sequence of CsHXK2 promoter."

Table 2

Key cis-acting elements in the CsHXK2 promoter of the tea plant"

Site name
-10PEHVPSBD TATTCT 叶绿体基因表达; 生理节律; 光调节。
Chloroplast gene expression; circadian rhythms; light regulation.
ARFAT TGTCTC ARF (生长素响应因子)结合位点; 生长素信号。
ARF (auxin response factor) binding site; auxin signaling.
ARR1AT NGATT ARR1结合元件; 细胞分裂素响应元件。
ARR1-binding element; cytokinin-responsive element.
BOXLCOREDCPAL ACCWWCC MYB结合位点; 苯丙氨酸解氨酶基因; 激发处理; UV-B辐射; 稀释效应。
MYB binding site; phenylalanine ammonia-lyase gene; elicitor treatment; UV-B irradiation; dilution effect.
Mesophyll-specific gene expression.
CANBNNAPA CNAACAC 胚和胚乳特异转录元件; 种子; 贮藏蛋白。
Element required for embryo and endosperm-specific transcription; seed; storage protein.
Flowering time.
Heat shock element.
CURECORECR GTAC 铜响应元件; 氧响应元件。
Copper-responsive element; oxygen-response element.
DOFCOREZM AAAG Dof蛋白核心结合位点; 碳代谢调控。
Core site required for binding of Dof proteins; regulating the carbon metabolism.
ELRECOREPCRP1 TTGACC 激发响应元件; WRKY结合位点; 水杨酸/病菌/创伤诱导信号。
Elicitor responsive element; WRKY protein binding site; salicylic acid/pathogen/wound- induced signaling.
ERELEE4 AWTTCAAA 乙烯响应元件; 衰老。
Ethylene responsive element; senescence.
GARE1OSREP1 TAACAGA 赤霉素响应元件; 种子。
Gibberellin-responsive element; seed.
Light-responsive element.
Light-responsive element.
Low-temperature-responsive element.
MYB1AT WAACCA MYB识别位点; 干旱/ABA响应元件。
MYB recognition site; dehydration/ABA-responsive element.
MYBCORE CNGTTR MYB结合位点; 干旱/水响应元件; 黄酮类生物合成。
MYB binding site; dehydration/water-responsive element; flavonoid biosynthesis.
MYBGAHV TAACAAA 赤霉素响应元件; MYB结合位点; α-淀粉酶。
Gibberellin response element; MYB binding site; α-amylase.
MYC recognition site; cold/dehydration/ABA-responsive element.
NTBBF1ARROLB ACTTTA 组织特异性表达与生长素诱导。
Tissue-specific expression and auxin induction.
Pollen specific expression.
RAV1AAT CAACA RAV1结合位点; 莲座叶和根。
RAV1 binding site; rosette leaves and roots.
RHERPATEXPA7 KCACGW 根毛特异性元件; 根; 毛。
Root hair-specific cis-element; root; hair.
Site name
SREATMSD TTATCC 糖抑制元件; 腋芽生长。
Sugar-repressive element; axillary bud outgrowth.
Sucrose responsive element.
TAAAGSTKST1 TAAAG 保卫细胞特异性基因表达。
Guard cell-specific gene expression.
WRKY binding site.
WBOXHVISO1 TGACT 糖响应元件; 糖信号; WRKY结合位点。
Sugar-responsive element; sugar signaling; WRKY binding site.

Fig. 2

Subcellular localization of CsHXK2 protein in the tea plant A-C and H-K: 35S::sGFP; D-G: 35S::CsHXK2::sGFP; L-O: 35S::CsHXK2-cTP::sGFP. A, D, H, L: GFP green fluorescent signal; E: chlorophyll autofluorescence; I, M: RFP red fluorescent signal of nucleus; B, F, J, N: bright field; C, G, K, O: merged signal."

Fig. 3

Complementation of the hexokinase-deficient yeast triple mutant with CsHXK2 protein A: ddH2O; B: 2% galactose; C: 2% glucose; D: 2% fructose; E: electrophoretic detection of the transformed fragments in yeast."

Fig. 4

Tissue-specific expression patterns of CsHXK2 in the tea plant A: different tissues of ‘Longjing 43’ in spring; B: different tissues of ‘Longjing 43’ in autumn; C: different tissues of ‘Shuchazao’. Different letters on the column indicate significant difference between the data (P < 0.05)."

Fig. 5

Expression analysis of CsHXK2 under different treatments in the tea plant A: natural cold acclimation; B: 4℃ cold and 25℃ recovery; C: inoculation with Colletotrichum; D: 50 μmol L-1 exogenous GA3. Different letters on the column indicate significant difference between the data (P < 0.05). * indicates significant at the 0.05 probability level; *** indicates significant at the 0.001 probability level."

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