Cloning of CsOLS Gene Promoter and Screening Upstream Transcription Factors in Hemp

doi:10.7668/hbnxb.20196830

Abstract

Abstract:

The cannabinoids in hemp have rich pharmacological activities.The olivetol synthase(CsOLS)gene of hemp is a key enzyme that catalyzes the synthesis of olivetolic acid(OA),a precursor substance of cannabinoids.To further investigate the expression regulation of the CsOLS gene,the promoter sequence of the CsOLS gene(CsOLSp)was cloned.Its transcriptional initiation site(TSS)and cis-elements were analysed through bioinformatics.The truncated fragments about 500 bp of CsOLSp,namely CsOLSp1,CsOLSp2,and CsOLSp3,were cloned and constructed into yeast one-hybrid bait vectors,followed by transformation into yeast cells and autoactivation assays.A cDNA library constructed from female hemp flowers and leaves was used for yeast one-hybrid screening.Candidate proteins obtained from the screening were subjected to one-to-one interaction validation,domain analysis,and molecular docking analysis.The results showed that the 1 500 bp promoter sequence of CsOLS gene was successfully cloned,which contained multiple hormone-responsive elements,light-responsive elements,and transcription factor binding sites.Autoactivation assays indicated that CsOLSp1 and CsOLSp3 were suitable for subsequent yeast library screening.Through primary and secondary screening,the numbers of proteins interacting with CsOLSp1 and CsOLSp3 were determined to be 5 and 7 respectively.One transcription factor was obtained from each,namely CsAHL14 and CsAHL15.One-to-one interaction assays confirmed that CsAHL14 interacted with CsOLSp1,while CsAHL15 interacted with CsOLSp3.Domain analysis revealed that CsAHL14 belonged to class B of the AHL family,whereas CsAHL15 belonged to class A.Molecular docking analysis further demonstrated that CsAHL14 exhibited strong binding affinity to the TTATTTTTTAT sequence.It was speculated that the transcription factor CsAHL14 might participate in the expression regulation of the CsOLS gene by specifically binding to the TTATTTTTTAT cis-acting element in the CsOLSp promoter,and play an important role in the cannabinoid biosynthesis process of hemp.

Key words: Hemp, CsOLS gene, Promoter, Yeast library screening, Transcription factor

CLC Number:

ZHAO Yan, YANG Jiang, SANG Yingying, ZHAI Ying, MA Tianyi, ZHANG Meijuan, LI Shanshan. Cloning of CsOLS Gene Promoter and Screening Upstream Transcription Factors in Hemp[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(5): 83-91. doi: 10.7668/hbnxb.20196830.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20196830

http://www.hbnxb.net/EN/Y2026/V41/I5/83

Figures/Tables 12

Fig.1 Truncated schematic diagram of CsOLSp

Fig.2 PCR amplification of the CsOLSp sequence and double restriction enzyme digestion of the recombinant plasmid M.DNA Marker 15000;1.PCR amplification product of the CsOLSp promoter;2.The double restriction enzyme digestion of the recombinant plasmid pMD18-T-CsOLSp.

Fig.3 Analysis of the TSS and cis-elements of the CsOLSp sequence A.Prediction of the TSS,with the +1 position indicated;B.Prediction of cis-elements excluding AT-rich elements;C.Prediction of AT-rich cis-elements.

Fig.4 PCR amplification of CsOLSp1,CsOLSp2 and CsOLSp3 fragments and double-enzyme digestion analysis of recombinant plasmids M.DNA Marker 15000;1,3,5.The PCR amplification products of the CsOLSp1,CsOLSp2,and CsOLSp3 fragments respectively;2,4,6.The double-enzyme digestion identification of the recombinant plasmids pAbAi-CsOLSp1,pAbAi-CsOLSp2,and pAbAi-CsOLSp3 respectively.

Fig.5 PCR identification of bait strains M.DNA Marker 2000;1—3.Yeast colony PCR of pAbAi-CsOLSp1,pAbAi-CsOLSp2,and pAbAi-CsOLSp3.

Fig.6 Self-activation assay of bait constructs p53-AbAi.The positive control strain Y1HGold[p53-AbAi];pAbAi-CsOLSp1,pAbAi-CsOLSp2,pAbAi-CsOLSp3.The bait strains of Y1HGold[pAbAi-CsOLSp1],Y1HGold[pAbAi-CsOLSp2],Y1HGold[pAbAi-CsOLSp3].

Tab.1 Result of yeast library screening

阳性克隆 Positive clone	同源基因登录号 Homologous gene accession number		功能注释 Functional annotation	出现次数 Occurrence frequency
CsOLSp1	1	XM_030637583.2	汉麻 L-抗坏血酸过氧化物酶2	17
	2	XM_030648159.2	汉麻 AT-hook基序核定位蛋白14	2
	3	XM_030637581.2	汉麻未表征蛋白LOC115709467	7
	4	XM_030626708.2	汉麻放氧增强蛋白2	1
	5	XM_030633900.2	汉麻未表征蛋白LOC115706300	1
CsOLSp3	1	XM_030624460.2	汉麻磷脂酰肌醇:神经酰胺肌醇磷酸转移酶1	5
	2	XM_030637932.2	汉麻ATP合酶O亚基	1
	3	XR_004012803.2	汉麻未表征非编码RNA	1
	4	XM_030634175.2	汉麻叶绿素a/b结合蛋白CP26	1
	5	XM_030643705.2	汉麻短链脱氢酶ptmH	1
	6	XM_030637842.2	汉麻含五肽重复序列蛋白At1g80270	1
	7	XM_061103180.1	汉麻 AT-hook 基序核定位蛋白15	1

Fig.7 PCR amplification of CsAHL14 and CsAHL15 fragments and double-enzyme digestion analysis of recombinant plasmids M.DNA Marker 15000;1,3.The PCR amplification products of the CsAHL14 and CsAHL15 fragments respectively;2,4.The double-enzyme digestion identification of the recombinant plasmids pGADT7-CsAHL14 and pGADT7-CsAHL15 respectively.

Fig.8 One-to-one interaction validation assay p53-AbAi + pGADT7-53.The positive control;pAbAi-CsOLSp1+pGADT7 and pAbAi-CsOLSp3+pGADT7.The negative controls.

Fig.9 Domain analysis of the AHL family in hemp

Fig.10 Molecular docking analysis of CsAHL14 and CsAHL15 with DNA motifs A.Overall structure of the CsAHL14-DNA complex;B.Binding mode of CsAHL14 with the TTATTTTTTAT motif;C.Overall structure of the CsAHL15-DNA complex;D.Binding mode of CsAHL15 with the TAAATAAATATATT motif.

Tab.2 Docking results for proteins and nucleic acid sequences

蛋白质 Protein	核酸 Nucleic acid	蛋白质结合位点 Binding site of protein	核酸结合位点 Binding site of nucleic acid	保守基序 Conserved motifs	ipTM
CsAHL14	CsOLSp1	ARG-81、ARG-83、ARG-85、LYS-88	+T4、+T6、+T8、-T9	TTATTTTTTAT	0.90
		ARG-83、ARG-85、ARG-87、LYS-88	+A7、-T6、-A7、-T9、-T10、-T11	AAAAATACATAC	0.88
		ARG-81、ARG-83、ARG-85、LYS-88	+T8、-A5、-T6、-T8、-T9、-T10	TAAAAAATAAAA	0.87
		ARG-81、ARG-83、ARG-85、LYS-88	+T6、-A7、-T8、-T10、-T12	AAAAATATTAAT	0.83
		ARG-83、GLY-84、ARG-85、LYS-88	+A2、+T6、-A3、-A5、-T6、-T7	GAATATTA	0.80
CsAHL15	CsOLSp3	ARG-104、ARG-105、ARG-107、ARG-109、GLY-112	+A10、+T11、-T7、-A10、-T11	TAAATAAATATATT	0.69
		ARG-109、PRO-110、GLY-112	-T8、-T7	AAATATTA	0.65
		GLY-112、ASN-115	-T7、-A6	AATATATATA	0.64

References 36

[1]	Sun X P. Research progress on cannabinoids in Cannabis(Cannabis sativa L.) in China[J]. Molecules, 2023, 28(9):3806.doi:10.3390/molecules28093806. URL
[2]	Zhang Z P, Wang Z, Lu J X, Yan S, He L Q, Wang P P, Qin C, Ren W C, Xu J, Wu J L, Liu X B, Ma W. In silico genome-wide analysis of homeodomain-leucine zipper transcription factors in Cannabis sativa L.[J]. Heliyon, 2024, 10(7):e28045.doi:10.1016/j.heliyon.2024.e28045. URL
[3]	Deguchi M, Dhir S, Potlakayala S, Dhir S, Curtis W R, Rudrabhatla S. In planta female flower agroinfiltration alters the cannabinoid composition in industrial hemp(Cannabis sativa L.)[J]. Frontiers in Plant Science, 2022, 13:921970.doi:10.3389/fpls.2022.921970. URL
[4]	刘永蓓, 张小雨. 工业大麻EMS突变体及大麻素合成相关基因表达分析[J]. 植物遗传资源学报, 2025, 26(2):394-403.doi:10.13430/j.cnki.jpgr.20240430002.
	Liu Y B, Zhang X Y. Construction of the EMS mutagenesis library for industrial hemp and expression of genes related to cannabinoid biosynthesis[J]. Journal of Plant Genetic Resources, 2025, 26(2):394-403.
[5]	Tan Z G, Clomburg J M, Gonzalez R. Synthetic pathway for the production of olivetolic acid in Escherichia coli[J]. ACS Synthetic Biology, 2018, 7(8):1886-1896.doi:10.1021/acssynbio.8b00075. URL
[6]	Zhang Y F, Guo J L, Gao P Z, Yan W, Shen J F, Luo X Z, Keasling J D. Development of an efficient yeast platform for cannabigerolic acid biosynthesis[J]. Metabolic Engineering, 2023, 80:232-240.doi:10.1016/j.ymben.2023.10.004. pmid: 37890610
[7]	朱雪雯, 米要磊, 孟祥霄, 陈伟强, 王思凡, 曹雪, 陈士林, 徐志超, 苏畅, 孙伟, 万会花. 汉麻聚酮合酶基因家族成员鉴定与表达分析[J]. 中草药, 2023, 54(3):886-897.doi:10.7501/j.issn.0253-2670.2023.03.023.
	Zhu X W, Mi Y L, Meng X X, Chen W Q, Wang S F, Cao X, Chen S L, Xu Z C, Su C, Sun W, Wan H H. Identification and expression analysis of polyketide synthase gene family in Cannabis sativa[J]. Chinese Traditional and Herbal Drugs, 2023, 54(3):886-897.
[8]	Fu D W, Chen Y H, Gao F. Yeast one-hybrid screening for transcription factors of IbbHLH2 in purple-fleshed sweet potato[J]. Genes, 2023, 14(5):1042.doi:10.3390/genes14051042. URL
[9]	Ma W H, Kang X, Liu P, She K X, Zhang Y Y, Lin X R, Li B, Chen Z Z. The NAC-like transcription factor CsNAC7 positively regulates the caffeine biosynthesis-related gene yhNMT1 in Camellia sinensis[J]. Horticulture Research, 2022, 9(1):3653-3665.doi:10.1093/hr/uhab046.
[10]	Peng Y, Guo X Q, Fan Y W, Liu H, Sun L Q, Liu D, Li H, Wang X, Guo H L, Lu H. Systematical characterization of the AT-Hook gene family in Juglans regia L.and the functional analysis of the JrAHL2 in flower induction and hypocotyl elongation[J]. International Journal of Molecular Sciences, 2023, 24:7244-7263.doi:10.3390/ijms24087244. URL
[11]	Peng Y, Guo X Q, Fan Y W, Liu H, Sun L Q, Liu D, Li H, Wang X, Guo H L, Lu H. Identifying a cis-element in PtoCP1 promoter for efficiently controlling constitutive gene expression in Populus tomentosa[J]. PeerJ, 2024, 12:e18292.doi:10.7717/peerj.18292. URL
[12]	张晓红, 许佩阳, 闫绪, 张贝贝, 于婵婵. 棉花GhFUL1基因及其启动子的克隆和功能分析[J]. 华北农学报, 2023, 38(5):60-68.doi:10.7668/hbnxb.20193992.
	Zhang X H, Xu P Y, Yan X, Zhang B B, Yu C C. Cloning and functional analysis of GhFUL1 gene and its promoter in cotton[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(5):60-68.
[13]	Jia H L, Wang X M, Shi Y H, Wu X M, Wang Y Q, Liu J N, Fang Z H, Li C Y, Dong K H. Overexpression of Medicago sativa LEA4-4 can improve the salt,drought,and oxidation resistance of transgenic Arabidopsis[J]. PLoS One, 2020, 15(6):e0234085.doi:10.1371/journal.pone.0234085. URL
[14]	Diao P F, Sun H Y, Bao Z, Li W X, Niu N, Li W M, Wuriyanghan H. Expression of an antiviral gene GmRUN1 from soybean is regulated via intron-mediated enhancement(IME)[J]. Viruses, 2021, 13(10):2032-2046.doi:10.3390/v13102032. URL
[15]	Liu W J, Wang Y C, Yu L, Jiang H Y, Guo Z W, Xu H F, Jiang S H, Fang H C, Zhang J, Su M Y, Zhang Z Y, Chen X L, Chen X S, Wang N. MdWRKY11 participates in anthocyanin accumulation in Red-Fleshed apples by affecting MYB transcription factors and the photoresponse factor MdHY5[J]. Journal of Agricultural and Food Chemistry, 2019, 67(32):8783-8793.doi:10.1021/acs.jafc.9b02920. pmid: 31310107
[16]	Liao B J, Liang P X, Tong L, Lu L, Lu Y, Zheng R H, Zheng X Y, Chen J H, Hao Z D. The role of Liriodendron Dof gene family in abiotic stress response[J]. Plants, 2024, 13(14):2009.doi:10.3390/plants13142009.
[17]	Liu S C, Wang Y, Shi M, Maoz I, Gao X K, Sun M H, Yuan T P, Li K L, Zhou W, Guo X H, Kai G Y. SmbHLH60 and SmMYC2 antagonistically regulate phenolic acids and anthocyanins biosynthesis in Salvia miltiorrhiza[J]. Journal of Advanced Research, 2022, 42:205-219.doi:10.1016/j.jare.2022.02.005. URL
[18]	Dahro B, Wang Y, Khan M, Zhang Y, Fang T, Ming R H, Li C L, Liu J H. Two AT-Hook proteins regulate A/NINV7 expression to modulate sucrose catabolism for cold tolerance in Poncirus trifoliata[J]. New Phytologist, 2022, 235(6):2331-2349.doi:10.1111/nph.18304. URL
[19]	Liu H, Dong S Y, Li M, Gu F W, Yang G L, Guo T, Chen Z Q, Wang J F. The Class Ⅲ peroxidase gene OsPrx30,transcriptionally modulated by the AT-hook protein OsATH1,mediates rice bacterial blight-induced ROS accumulation[J]. Journal of Integrative Plant Biology, 2021, 63(2):393-408.doi:10.1111/jipb.13040. URL
[20]	Xiong S X, Zeng Q Y, Hou J Q, Hou L L, Zhu J, Yang M, Yang Z N, Lou Y. The temporal regulation of TEK contributes to pollen wall exine patterning[J]. PLoS Genetics, 2020, 16(5):e1008807.doi:10.1371/journal.pgen.1008807. URL
[21]	Shi X W, Yang T, Ren M F, Fu J, Bai J, Cui H C. AT-hook motif nuclear localized transcription factors function redundantly in promoting root growth through modulation of redox homeostasis[J]. The Plant Journal, 2024, 120(1):199-217.doi:10.1111/tpj.16981. pmid: 39136690
[22]	Wang M, Chen B W, Zhou W, Xie L N, Wang L S, Zhang Y L, Zhang Q Z. Genome-wide identification and expression analysis of the AT-hook Motif Nuclear Localized gene family in soybean[J]. BMC Genomics, 2021, 22(1):361.doi:10.1186/s12864-021-07687-y. pmid: 34006214
[23]	Jia Q S, Zhu J, Xu X F, Lou Y, Zhang Z L, Zhang Z P, Yang Z N. Arabidopsis AT-hook protein TEK positively regulates the expression of Arabinogalactan proteins for nexine formation[J]. Molecular Plant, 2015, 8(2):251-260.doi:10.1016/j.molp.2014.10.001. URL
[24]	Liu Y Y, Zhu P P, Cai S, Haughn G, Page J E. Three novel transcription factors involved in cannabinoid biosynthesis in Cannabis sativa L.[J]. Plant Molecular Biology, 2021, 106(1):49-65.doi:10.1007/s11103-021-01129-9.
[25]	Fayaz M, Angmo T, Katoch K, Majeed A, Kundan M, Wajid M A, Pal K, Misra P. The promoter regions of CBDAS and PT genes of cannabinoid biosynthesis in Cannabis sativa respond to phytohormones and stress-related signals[J]. Planta, 2025, 261(6):135.doi:10.1007/s00425-025-04709-x.
[26]	Hiratsuka T, Makita Y, Yamamoto Y Y. Sequence-based evaluation of promoter context for prediction of transcription start sites in Arabidopsis and rice[J]. Scientific Reports, 2022, 12:6976.doi:10.1038/s41598-022-11169-w. pmid: 35484393
[27]	Haberle V, Stark A. Eukaryotic core promoters and the functional basis of transcription initiation[J]. Nature Reviews Molecular Cell Biology, 2018, 19(10):621-637.doi:10.1038/s41580-018-0028-8. pmid: 29946135
[28]	Favero D S, Kawamura A, Shibata M, Takebayashi A, Jung J H, Suzuki T, Jaeger K E, Ishida T, Iwase A, Wigge P A, Neff M M, Sugimoto K. AT-hook transcription factors restrict petiole growth by antagonizing PIFs[J]. Current Biology, 2020, 30(8):1454-1466.e6.doi:10.1016/j.cub.2020.02.017. pmid: 32197081
[29]	Lou Y, Xu X F, Zhu J, Gu J N, Blackmore S, Yang Z N. The tapetal AHL family protein TEK determines nexine formation in the pollen wall[J]. Nature Communications, 2014, 5:3855.doi:10.1038/ncomms4855. pmid: 24804694
[30]	Karami O, Rahimi A, Mak P, Horstman A, Boutilier K, Compier M, van der Zaal B, Offringa R. An Arabidopsis AT-hook motif nuclear protein mediates somatic embryogenesis and coinciding genome duplication[J]. Nature Communications, 2021, 12(1):2508.doi:10.1038/s41467-021-22815-8. pmid: 33947865
[31]	Širl M, Šnajdrová T, Gutiérrez-Alanís D, Dubrovsky J G, Vielle-Calzada J P, Kulich I, Soukup A. At-hook motif nuclear localised protein 18 as a novel modulator of root system architecture[J]. International Journal of Molecular Sciences, 2020, 21(5):1886.doi:10.3390/ijms21051886.
[32]	Karami O, Rahimi A, Khan M, Bemer M, Hazarika R R, Mak P, Compier M, van Noort V, Offringa R. A suppressor of axillary meristem maturation promotes longevity in flowering plants[J]. Nature Plants, 2020, 6(4):368-376.doi:10.1038/s41477-020-0637-z. pmid: 32284551
[33]	Wong M M, Huang X J, Bau Y C, Verslues P E. AT Hook-Like 10 phosphorylation determines ribosomal RNA processing 6-like 1(RRP6L1)chromatin association and growth suppression during water stress[J]. Plant,Cell & Environment, 2024, 47(1):24-37.doi:10.1111/pce.14725. URL
[34]	Chen Y, Luo M Y, Li P, Zhao Q, Chao L, Jiao Y J, Tian S Y, Tian X Y, Ye S Y, Yuan Z, Hu Y L, Bai Y Q, Wan Y, Wang D J, Chen W Y, Yao X Z, Lu L T. CsAHL20 negatively regulates epi-catechins biosynthesis under drought stress in Camellia sinensis[J]. Plant Science, 2025, 359:112613.doi:10.1016/j.plantsci.2025.112613. URL
[35]	Kim J, Kim D G, Ha T H, Kim W J, Ryu J, Kim J B, Kim S H. Effect of hormonal treatments on cannabinoid content levels in female hemp(Cannabis sativa L.) inflorescences[J]. International Journal of Molecular Sciences, 2025, 26(7):3445-3458.doi:10.3390/ijms26073445. URL
[36]	Zhang Y, Song T T, Xia Y X. Transcription factor MaAP-1 regulates conidiation patterns via YAP domain binding to the MaPom1 promoter in Metarhizium acridum:implications for enhancing fungal biocontrol efficiency[J]. Pesticide Biochemistry and Physiology, 2025, 214:106587.doi:10.1016/j.pestbp.2025.106587. URL

Please choose a citation manager

Content to export