工业大麻转录因子CsMYB12抗旱功能的研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-0782

摘要/Abstract

摘要：

目的 MYB转录因子家族成员在植物中数量众多，它们在调控植物的生长发育及适应外界环境胁迫中发挥重要作用。工业大麻作为经济作物，用途广泛，具有极大的开发利用潜力。揭示工业大麻转录因子基因CsMYB12的抗旱功能，为工业大麻品种的抗旱性改良及产量提升奠定基础。方法通过实时荧光定量PCR检测CsMYB12在干旱胁迫下的表达；克隆CsMYB12并对其进行生物信息学分析；通过酵母转录激活试验检测CsMYB12转录激活活性；构建CsMYB12植物表达载体转化烟草，并对转基因烟草的抗旱性进行鉴定。结果干旱胁迫可以显著诱导CsMYB12上调表达。CsMYB12开放阅读框序列长1 560 bp，编码519个氨基酸。CsMYB12蛋白分子量为5.81 kD，等电点为4.94。CsMYB12蛋白含有2个SANT结构域，是一个典型的R2R3-MYB转录因子。CsMYB12在酵母细胞中具有转录激活活性。经鉴定，共获得6株CsMYB12转基因烟草植株。干旱胁迫及复水处理后，CsMYB12转基因烟草的表现优于野生型烟草。干旱胁迫后，与野生型烟草相比，CsMYB12转基因烟草中的渗透调节物质含量、相对含水量和抗氧化酶活性增加，电解质渗透率和丙二醛含量下降。CsMYB12转基因烟草中抗逆相关基因（NtLTP、NtOsmotin、NtLEA5、NtERD10B和NtCSD）的表达量显著升高。结论 CsMYB12在烟草中的异源超表达提高了转基因烟草的抗旱性。

关键词: 工业大麻, 转录因子, MYB, 转基因烟草, 抗旱性

Abstract:

Objective There are numerous MYB transcription factor family members in plants. They play a crucial role in regulating the growth and development of plants as well as in enabling plants to adapt to external environmental stress. Industrial hemp (Cannabis sativa L.), as an economic crop, has a wide range of applications and holds great potential for development and utilization. Revealing the drought resistance function of the transcription factor gene CsMYB12 in industrial hemp may lay a foundation for improving the drought resistance and yield of industrial hemp varieties. Method The expression of CsMYB12 under drought stress was detected by real-time fluorescent quantitative PCR. CsMYB12 was cloned and subjected to bioinformatics analysis. The transcriptional activation activity of CsMYB12 was detected through yeast transcriptional activation test. The CsMYB12 plant expression vector was constructed and transformed into tobacco. The resistance of CsMYB12 transgenic tobacco to drought was identified. Result The expressions of CsMYB12 were significantly induced by drought stress. The open reading frame of CsMYB12 was 1 560 bp and encoded 519 amino acids. The molecular weight of the CsMYB12 protein was 5.81 kD and its isoelectric point was 4.94. The CsMYB12 protein contained two SANT domains and it was a typical R2R3-MYB transcription factor. CsMYB12 had transcriptional activation activity in yeast cells. Six CsMYB12 transgenic tobacco plants were identified. After drought stress and rehydration treatment, the performance of CsMYB12 transgenic tobacco was superior to that of wild-type tobacco. After drought stress, the content of osmotic regulatory substances, relative water content and antioxidant enzyme activities in CsMYB12 transgenic tobacco increased, while the electrolyte leakage rate and malondialdehyde content decreased compared with the wild-type tobacco. The expressions of stress-related genes (NtLTP, NtOsmotin, NtLEA5, NtERD10B and NtCSD) in CsMYB12 transgenic tobacco significantly increased. Conclusion The heterologous overexpression of CsMYB12 in tobacco enhances the resistance of transgenic tobacco to drought.

Key words: industrial hemp, transcription factor, MYB, transgenic tobacco, resistance to drought

翟莹, 高双, 计俊杰, 于海伟, 赵艳, 马天意, 张梅娟, 李珊珊. 工业大麻转录因子CsMYB12抗旱功能的研究[J]. 生物技术通报, 2026, 42(6): 294-303.

ZHAI Ying, GAO Shuang, JI Jun-jie, YU Hai-wei, ZHAO Yan, MA Tian-yi, ZHANG Mei-juan, LI Shan-shan. Study on the Drought Resistance Function of the Transcription Factor CsMYB12 Gene in Industrial Hemp[J]. Biotechnology Bulletin, 2026, 42(6): 294-303.

图/表 9

表1 PCR引物序列

Table 1 PCR primer sequences

基因 Gene	GenBank登录号 GenBank accession number	正向引物 Forward primer （5′‒3′）	反向引物 Reverse primer （5′‒3′）	用途 Use
CsMYB12	XM030631399	GAAGAAGGAAGTGGTGGTTGTGG	AGCAGTAGCAGGAACAGGAGATAC	RT-qPCR
CsEF1a	JP452083	TGTTTTGCACGGATCAGTTTG	AATGCCGACCGCTACAGTTC	RT-qPCR
CsMYB12	XM030631399	ATGGGTAGGGCTCCTTGCTG	TCAGGATAGAAGCCATGCAACC	PCR
NtActin	AB158612	TTGCTGGTCGTGATCTTACTGATTG	CAGTCTCCAACTCTTGCTCATAGTC	RT-qPCR
NtLTP	AB625593	TTAAGGGCATTGATATGGGCAACG	GTGGAGGGACTGATCTTGTAGGG	RT-qPCR
NtOsmotin	M29279	CTCCTTGCCTTGGTGACTT	ACCGCCTATGGGTGTCG	RT-qPCR
NtLEA5	AF053076	TGGCTCGCTCTTTCTCTAACTCC	CTCACTCCACCTGGCACACTAG	RT-qPCR
NtERD10B	AB049336	TCCCATTCGTCAAACCG	CCCACCAAGTATGCCAGT	RT-qPCR
NtCSD	EU123521	CCATTACCGACAAGCAGATTCCTC	CAACCCTTCCACCAGCATTTCC	RT-qPCR

图1 干旱胁迫下CsMYB12的表达不同字母表示差异显著（P0.05）。下同

Fig. 1 Expressions of CsMYB12 gene under drought stressDifferent letters indicate significant differences (P0.05). The same below

图2 CsMYB12核苷酸及其编码蛋白氨基酸序列蓝色代表SANT结构域；*代表终止密码子

Fig. 2 Nucleotide of CsMYB12 and amino acid sequences of its encoded proteinBlue refers to the HSANT domain; * refers to the termination codon

图3 CsMYB12转录激活活性分析

Fig. 3 Transcriptional activating activity of CsMYB12

图4 CsMYB12植物过表达载体的构建及转基因烟草鉴定A：CsMYB12重组载体的构建；B：T0代转基因烟草的PCR检测；C：T0代转基因烟草的RT-qPCR检测。M：DL2000分子量标记物；1：pRI101-CsMYB12质粒双酶切；2：pRI101-CsMYB12农杆菌菌液PCR；+：pRI101-CsMYB12质粒；WT：野生型烟草；OE1-OE6：CsMYB12转基因烟草。下同

Fig. 4 Construction of CsMYB12 plant overexpression vector and identification of transgenic tobaccoA: Construction of CsMYB12 recombinant vector. B: PCR detection of T0 generation transgenic tobacco. C: RT-qPCR detection of T0 generation transgenic tobacco. M: DL2000 marker for molecular weight. 1: Double enzyme digestion of pRI101-CsMYB12 plasmid. 2: Agrobacterium tumefaciens PCR of PRI101-CsMYB12. +: PRI101-CsMYB12 plasmid. WT: Wild-type tobacco. OE1-OE6: CsMYB12 transgenic tobacco. The same below

图5 CsMYB12转基因烟草在干旱及复水处理下的表型A：停止浇水前；B：停止浇水20 d；C：停止浇水34 d；D：恢复浇水5 d

Fig. 5 Phenotype of CsMYB12 transgenic tobacco under drought and rehydration treatmentA: Before stop watering. B: Stop watering for 20 d. C: Stop watering for 34 d. D: Rehydration for 5 d

图6 CsMYB12转基因烟草干旱胁迫下的生理生化指标

图7 CsMYB12转基因烟草干旱胁迫下叶片DAB和NBT染色A：DAB染色；B：NBT染色

Fig. 7 DAB and NBT staining of CsMYB12 transgenic tobacco leaves under drought stressA: DAB staining. B: NBT staining

图8 CsMYB12转基因烟草胁迫相关基因的表达*和**分别表示在P0.05和P0.01水平上差异显著

Fig. 8 Expressions of stress-related genes in CsMYB12 transgenic tobacco* and ** refer to significant difference at P0.05 and P0.01, respectively

参考文献 52

[1]	Huang GT, Ma SL, Bai LP, et al. Signal transduction during cold, salt, and drought stresses in plants [J]. Mol Biol Rep, 2012, 39(2): 969-987.
[2]	Bassolino L, Buti M, Fulvio F, et al. In silico identification of MYB and bHLH families reveals candidate transcription factors for secondary metabolic pathways in Cannabis sativa L. [J]. Plants, 2020, 9(11): 1540.
[3]	Gao CS, Xin PF, Cheng CH, et al. Diversity analysis in Cannabis sativa based on large-scale development of expressed sequence tag-derived simple sequence repeat markers [J]. PLoS One, 2014, 9(10): e110638.
[4]	Andre CM, Hausman JF, Guerriero G. Cannabis sativa: the plant of the thousand and one molecules [J]. Front Plant Sci, 2016, 7: 19.
[5]	潘凌云, 马家冀, 李建民, 等. 植物盐胁迫应答转录因子的研究进展 [J]. 生物工程学报, 2022, 38(1): 50-65.
	Pan LY, Ma JJ, Li JM, et al. Advances of salt stress-responsive transcription factors in plants [J]. Chinese Journal of Biotechnology, 2022, 38(1): 50-65.
[6]	王彬, 陈敏氡, 林亮, 等. 植物干旱胁迫的信号通路及相关转录因子研究进展 [J]. 西北植物学报, 2020, 40(10): 1792-1806.
	Wang B, Chen MD, Lin L, et al. Signal pathways and related transcription factors of drought stress in plants [J]. Acta Botanica Boreali-Occidentalia Sinica, 2020, 40(10): 1792-1806.
[7]	Paz-Ares J, Ghosal D, Wienand U, et al. The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators [J]. EMBO J, 1987, 6(12): 3553-3558.
[8]	Pratyusha DS, Sarada DVL. MYB transcription factors-master regulators of phenylpropanoid biosynthesis and diverse developmental and stress responses [J]. Plant Cell Rep, 2022, 41(12): 2245-2260.
[9]	Seven M, Akdemir H. DOF, MYB and TCP transcription factors: Their possible roles on barley germination and seedling establishment [J]. Gene Expr Patterns, 2020, 37: 119116.
[10]	Ogata K, Kanei-Ishii C, Sasaki M, et al. The cavity in the hydrophobic core of Myb DNA-binding domain is reserved for DNA recognition and trans-activation [J]. Nat Struct Biol, 1996, 3(2): 178-187.
[11]	何锐, 兴旺, 刘大丽, 等. MYB调控靶基因参与植物应答重金属胁迫的研究进展 [J]. 植物遗传资源学报, 2025, 26(3): 419-430.
	He R, Xing W, Liu DL, et al. Research progress of MYB regulated target genes involved in response to heavy metal stress [J]. Journal of Plant Genetic Resources, 2025, 26(3): 419-430.
[12]	李志强, 罗正乾, 徐琳黎, 等. 基于T2T基因组鉴定大豆R2R3-MYB基因家族及干旱和盐胁迫下的表达分析 [J]. 生物技术通报, 2025, 41(5): 141-152.
	Li ZQ, Luo ZQ, Xu LL, et al. Identification of the soybean R2R3-MYB gene family based on the T2T genome and their expression analysis under drought and salt stress [J]. Biotechnology Bulletin, 2025, 41(5): 141-152.
[13]	Fang Q, Wang XQ, Wang HY, et al. The poplar R2R3 MYB transcription factor PtrMYB94 coordinates with abscisic acid signaling to improve drought tolerance in plants [J]. Tree Physiol, 2020, 40(1): 46-59.
[14]	Lv KW, Wei HR, Liu GF. A R2R3-MYB transcription factor gene, BpMYB123, regulates BpLEA14 to improve drought tolerance in Betula platyphylla [J]. Front Plant Sci, 2021, 12: 791390.
[15]	Wu YQ, Li TT, Cheng ZY, et al. R2R3-MYB transcription factor PlMYB108 confers drought tolerance in herbaceous peony (Paeonia lactiflora Pall.) [J]. Int J Mol Sci, 2021, 22(21): 11884.
[16]	Zhao HX, Yao PF, Zhao JL, et al. A novel R2R3-MYB transcription factor FtMYB22 negatively regulates salt and drought stress through ABA-dependent pathway [J]. Int J Mol Sci, 2022, 23(23): 14549.
[17]	Chen YN, Feng PP, Zhang XW, et al. Silencing of SlMYB50 affects tolerance to drought and salt stress in tomato [J]. Plant Physiol Bioch, 2022, 193: 139-152.
[18]	van Bakel H, Stout JM, Cote AG, et al. The draft genome and transcriptome of Cannabis sativa [J]. Genome Biol, 2011, 12(10): R102.
[19]	Amaducci S, Zatta A, Pelatti F, et al. Influence of agronomic factors on yield and quality of hemp (Cannabis sativa L.) fibre and implication for an innovative production system [J]. Field Crop Res, 2008, 107(2): 161-169.
[20]	Žydelis R, Herbst M, Weihermüller L, et al. Yield potential and factor influencing yield gap in industrial hemp cultivation under nemoral climate conditions [J]. Eur J Agron, 2022, 139: 126576.
[21]	Guo R, Guo HY, Zhang QY, et al. Evaluation of reference genes for RT-qPCR analysis in wild and cultivated Cannabis [J]. Biosci Biotech Bioch, 2018, 82(11): 1902-1910.
[22]	翟莹. 大豆ERF转录因子GmERF6和GmERF7的克隆与功能鉴定 [D]. 长春: 吉林大学, 2012.
	Zhai Y. Isolation and characterization of ERF transcription factors, GmERF6 and GmERF7, from soybean (Glycine max L.) [D]. Changchun: Jilin University, 2012.
[23]	Hoekema A, Hirsch PR, Hooykaas PJJ, et al. A binary plant vector strategy based on separation of Vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid [J]. Nature, 1983, 303(5913): 179-180.
[24]	翟莹, 李铭杨, 张军, 等. 异源表达大豆转录因子GmNF-YA19提高转基因烟草抗旱性 [J]. 生物技术通报, 2023, 39(5): 224-232.
	Zhai Y, Li MY, Zhang J, et al. Heterologous expression of soybean transcription factor GmNF-YA19 improves drought resistance of transgenic tobacco [J]. Biotechnology Bulletin, 2023, 39(5): 224-232.
[25]	Qiu S, Zhang J, He JQ, et al. Overexpression of GmGolS2-1, a soybean galactinol synthase gene, enhances transgenic tobacco drought tolerance [J]. Plant Cell Tiss Org, 2020, 143(3): 507-516.
[26]	Shao HB, Liang ZS, Shao MG. Osmotic regulation of 10 wheat (Triticum aestivum L.) genotypes at soil water deficits [J]. Colloid Surface B, 2006, 47(2): 132-139.
[27]	Zhai Y, Wang Y, Li YJ, et al. Isolation and molecular characterization of GmERF7, a soybean ethylene-response factor that increases salt stress tolerance in tobacco [J]. Gene, 2013, 513(1): 174-183.
[28]	Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water-stress studies [J]. Plant Soil, 1973, 39(1): 205-207.
[29]	Sun XD, Lian HF, Liu XC, et al. The garlic NF-YC gene, AsNF-YC8 positively regulates non-ionic hyperosmotic stress tolerance in tobacco [J]. Protoplasma, 2017, 254(3): 1353-1366.
[30]	田琴, 刘奎, 吴翔纬, 等. 转录因子VcMYB17调控蓝莓抗旱性的功能研究 [J]. 生物技术通报, 2025, 41(4): 198-210.
	Tian Q, Liu K, Wu XW, et al. Functional study of transcription factor VcMYB17 in regulating drought tolerance in blueberry [J]. Biotechnology Bulletin, 2025, 41(4): 198-210.
[31]	Boyer LA, Latek RR, Peterson CL. The SANT domain: a unique histone-tail-binding module [J]. Nat Rev Mol Cell Biol, 2004, 5(2): 158-163.
[32]	Hu XM, Liang ZH, Sun TX, et al. The R2R3-MYB transcriptional repressor TgMYB4 negatively regulates anthocyanin biosynthesis in tulips (Tulipa gesneriana L.) [J]. Int J Mol Sci, 2024, 25(1): 563.
[33]	Song PH, Yang RH, Jiao KB, et al. FvMYB108, a MYB gene from Fragaria vesca, positively regulates cold and salt tolerance of Arabidopsis [J]. Int J Mol Sci, 2024, 25(6): 3405.
[34]	Zhang L, Xu Y, Li YT, et al. Transcription factor CsMYB77 negatively regulates fruit ripening and fruit size in Citrus [J]. Plant Physiol, 2024, 194(2): 867-883.
[35]	Jiang LL, Chen JH, Qian JY, et al. The R2R3-MYB transcription factor ZeMYB32 negatively regulates anthocyanin biosynthesis in Zinnia elegans [J]. Plant Mol Biol, 2024, 114(3): 48.
[36]	乔景璇, 李林, 何汶珈, 等. 花生转录因子AhMYB86的生物信息学及表达模式分析 [J/OL]. 分子植物育种, 2023. .
	Qiao JX, Li L, He WJ, et al. Bioinformatic analysis and expression pattern of peanut transcription factor AhMYB86 [J/OL]. Molecular Plant Breeding, 2023. .
[37]	马乐. 低温胁迫下软枣猕猴桃AaMYB117基因的功能研究 [D]. 哈尔滨: 东北林业大学, 2023.
	Ma L. The function of AaMYB117 gene in Actinidiaarguta under low temperature stress[D]. Harbin: Northeast Forestry University, 2023.
[38]	方森. 苹果转录因子MhMYB4抗旱功能鉴定及其作用机制解析 [D]. 郑州: 河南农业大学, 2023.
	Fang S. Identification of drought resistance function of apple transcription factor MhMYB4 and its mechanism analysis [D]. Zhengzhou: Henan Agricultural University, 2023.
[39]	Cao ZH, Zhang SZ, Wang RK, et al. Genome wide analysis of the apple MYB transcription factor family allows the identification of MdoMYB121 gene confering abiotic stress tolerance in plants [J]. PLoS One, 2013, 8(7): e69955.
[40]	Yan DH, Fenning T, Tang S, et al. Genome-wide transcriptional response of Populus euphratica to long-term drought stress [J]. Plant Sci, 2012, 195: 24-35.
[41]	刘汉嶂. 蒙古栎R2R3-MYB基因家族鉴定及其与抗旱相关基因功能特性研究 [D]. 沈阳: 沈阳农业大学, 2023.
	Liu HZ. Identification of R2R3-MYB gene family of Quercus mongolica and functional characteristics of genes related to drought resistance [D]. Shenyang: Shenyang Agricultural University, 2023.
[42]	任明辉, 张雨蓬, 许涛, 等. 紫花苜蓿R2R3-MYB亚家族鉴定与干旱胁迫下的表达分析 [J]. 草地学报, 2023, 31(4): 972-983.
	Ren MH, Zhang YP, Xu T, et al. Identification and expression analyses of R2R3-MYB subfamily in alfalfa under drought stress [J]. Acta Agrestia Sinica, 2023, 31(4): 972-983.
[43]	王悦, 刘亚玲, 苑峰, 等. 蒺藜苜蓿MtMYB16基因克隆、表达及转录自激活分析 [J]. 中国草地学报, 2024, 46(03): 1-10.
	Wang Y, Liu YL, Yuan F, et al. Cloning, expression and transcriptional autoactivation analysis of MtMYB16 gene in Medicago truncatula [J]. Chinese Journal of Grassland, 2024, 46(03): 1-10.
[44]	徐子寅. 木薯MeMYB60转录因子低温胁迫相关的互作蛋白及下游基因的筛选与验证 [D]. 海口: 海南大学, 2023.
	Xu ZY. Screening and validation of interacting proteins and downstream genes related to low temperature stress of cassava MeMYB60 transcription factor[D]. Haikou: Hainan University, 2023.
[45]	杨宗燃. 二色补血草MYB转录因子LbMYB17的功能研究 [D]. 济南: 山东师范大学, 2024.
	Yang ZL. Functional study of MYB transcription factor LbMYB17 of Limonium bicolor [D]. Jinan: Shandong Normal University, 2024.
[46]	Maiti R, Satya P. Research advances in major cereal crops for adaptation to abiotic stresses [J]. Gm Crops Food, 2014, 5(4): 259-279.
[47]	Fu CL, Bian CY, Chen J, et al. LcMYB5, an R2R3-MYB family gene from Lonicera caerulea L., enhances drought and salt tolerance in transgenic tobacco and blue honeysuckle [J]. J Plant Physiol, 2025, 304: 154409.
[48]	Hu W, Huang C, Deng XM, et al. TaASR1, a transcription factor gene in wheat, confers drought stress tolerance in transgenic tobacco [J]. Plant Cell Environ, 2013, 36(8): 1449-1464.
[49]	Li SF, Zhang YX, Xin XB, et al. The osmotin-like protein gene PdOLP1 is involved in secondary cell wall biosynthesis during wood formation in poplar [J]. Int J Mol Sci, 2020, 21(11): 3993.
[50]	Yang Z, Mu Y, Wang Y, et al. Characterization of a novel TtLEA2 gene from Tritipyrum and its transformation in wheat to enhance salt tolerance[J]. Front Plant Sci, 2022, 13: 830848.
[51]	Zhuo CL, Wang T, Guo ZF, et al. Overexpression of MfPIP2-7 from Medicago falcata promotes cold tolerance and growth under NO₃ ^- deficiency in transgenic tobacco plants [J]. BMC Plant Biol, 2016, 16(1): 138.
[52]	Huo CS, He LS, Yu T, et al. The superoxide dismutase gene family in Nicotiana tabacum: genome-wide identification, characterization, expression profiling and functional analysis in response to heavy metal stress [J]. Front Plant Sci, 2022, 13: 904105.