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作物学报 ›› 2024, Vol. 50 ›› Issue (11): 2754-2763.doi: 10.3724/SP.J.1006.2024.44001

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

工业大麻腺毛发育基因CsMIXTA启动子克隆及功能分析

周智满1(), 张小雨1, 高峰2, 戴志刚1, 许英1, 程超华1, 杨泽茂1, 粟建光1, 唐蜻1,*()   

  1. 1中国农业科学院麻类研究所 / 种质资源研究室, 湖南长沙 410205
    2云南省工业大麻产业研究院, 云南昆明 650214
  • 收稿日期:2024-01-02 接受日期:2024-06-20 出版日期:2024-11-12 网络出版日期:2024-07-12
  • 通讯作者: *唐蜻, E-mail: qingtang1996@163.com
  • 作者简介:E-mail: 857033058@qq.com
  • 基金资助:
    财政部和农业农村部国家现代农业产业技术体系建设专项(麻类, CARS-16-E01);中国农业科学院创新工程项目(CAASASTIP-2017-IBFC01)

Cloning and functional analysis of promoter of CsMIXTA associated with development of glandular trichome in industrial hemp

ZHOU Zhi-Man1(), ZHANG Xiao-Yu1, GAO Feng2, DAI Zhi-Gang1, XU Ying1, CHENG Chao-Hua1, YANG Ze-Mao1, SU Jian-Guang1, TANG Qing1,*()   

  1. 1Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences / Laboratory of Germplasm Resources, Changsha 410205, Hunan, China
    2Yunnan Academy of Industrial Hemp, Kunming 650214, Yunnan, China
  • Received:2024-01-02 Accepted:2024-06-20 Published:2024-11-12 Published online:2024-07-12
  • Contact: *E-mail: qingtang1996@163.com
  • Supported by:
    China Agriculture Research System of MOF and MARA (Bast and Leaf Fiber Crops, CARS-16-E01);Science and Technology Innovation Project of the Chinese Academy of Agricultural Sciences(CAASASTIP-2017-IBFC01)

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摘要：

CsMIXTA在调控大麻雌性花器官中腺毛的发生和发育中发挥重要作用。为了研究CsMIXTA基因的表达调控, 对CsMIXTA基因上游2199 bp启动子序列进行了克隆。通过PlantCARE预测发现, 该启动子序列具有多个胁迫响应元件与光响应元件。根据预测的响应元件分布情况, 扩增获得5个5′端缺失的启动子片段。用克隆的启动子全长和缺失片段分别构建融合GUS基因的表达载体, 并将其瞬时转化到烟草叶片和工业大麻糖叶中。通过GUS染色观察发现, CsMIXTA启动子的核心区域位于-393~ -99 bp之间, 包含赤霉素响应元件TATC-box和转录起始元件TATA-box。通过LUC荧光素酶表达发现, CsMIXTA启动子的核心区域具有转录活性。研究还发现, CsMIXTA基因在工业大麻糖叶的腺毛中特异性表达。对启动子进行胁迫响应分析的结果显示, 低温、脱落酸(ABA)和赤霉素(GA3)都能增强该启动子的活性。这些结果为CsMIXTA基因调控机制研究奠定了重要基础。

关键词: 工业大麻, CsMIXTA启动子, 瞬时表达, GA3、ABA处理

Abstract:

CsMIXTA may play a crucial role in the morphology and development of glandular trichomes in cannabis female flowers. To investigate its regulatory mechanism, a 2199 bp promoter sequence of CsMIXTA was cloned. PlantCARE prediction identified multiple hormone response elements and stress response elements within this region. Based on this analysis, five 5′ end deletion fragments of the promoter with varying lengths were amplified. Six GUS gene expression vectors were constructed using the full-length promoter and the 5′ end deletion fragments, which were then transiently expressed in tobacco leaves and industrial hemp sugar leaves. GUS staining revealed that nucleotide positions -393 and -99 constituted the core region of the CsMIXTA promoter, containing the gibberellin response element TATC-box and the transcription initiation element TATA-box. The results also demonstrated that CsMIXTA is specifically expressed in glandular trichomes of industrial hemp. Promoter activity of the core region was confirmed by luciferase assay. Stress response analysis indicated that low temperature, abscisic acid (ABA), and gibberellin (GA3) enhanced promoter activity. These findings provide a crucial basis for further studies on the regulation of CsMIXTA.

Key words: industrial hemp, CsMIXTA promoter, transient expression, GA3 and ABA treatment

表1

本研究所用引物"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 用途
Usage
P-F TATAAGCATATTTTACAATTTTG 启动子全长克隆
Cloning of full-length promoters
P1-F CAACCTACCTTAATATCATA 缺失片段1克隆
Cloning of deletion fragment 1
P2-F ATGCACATCACTTCA 缺失片段2克隆
Cloning of deletion fragment 2
P3-F GAAATAACTTGAAGGCT 缺失片段3克隆
Cloning of deletion fragment 3
P4-F GAGCATAATATTGTAGA 缺失片段4克隆
Cloning of deletion fragment 4
P5-F CTCATATACTCTTTCTCT 缺失片段5克隆
Cloning of deletion fragment 4
P-R ATATATATATATATATGATCGATCT 启动子所有片段克隆
Cloning of all fragments of the promoter
P-JB-F ggctgcaggtcgacggatccTATAAGCATATTTTACAATTTTG 启动子全长构建pCAMBIA1391
Full-length promoter construct pCAMBIA1391
P1-JB-F ggctgcaggtcgacggatccCAACCTACCTTAATATCATA 缺失片段1构建pCAMBIA1391
Deletion fragment 1 construct pCAMBIA1391
P2-JB-F ggctgcaggtcgacggatccATGCACATCACTTCA 缺失片段2构建pCAMBIA1391
Deletion fragment 2 construct pCAMBIA1391
P3-JB-F ggctgcaggtcgacggatccGAAATAACTTGAAGGCT 缺失片段3构建pCAMBIA1391
Deletion fragment 3 construct pCAMBIA1391
P4-JB-F ggctgcaggtcgacggatccGAGCATAATATTGTAGA 缺失片段4构建pCAMBIA1391
Deletion fragment 4 construct pCAMBIA1391
P5-JB-F ggctgcaggtcgacggatccCTCATATACTCTTTCTCT 缺失片段5构建pCAMBIA1391
Deletion fragment 5 construct pCAMBIA1391
P-JB-R cttagaattcccggggatccATATATATATATATATGATCGATCT 启动子所有片段构建pCAMBIA1391
All fragments construct pCAMBIA1391
P4-NCJB-F agtggtctctgtccagtcctGAGCATAATATTGTAGA 缺失片段4构建pNC-Green-LUC
Deletion fragment 4 construct pNC-Green-LUC
P-NCJB-R ggtctcagcagaccacaagtATATATATATATATATGATCGATCT 缺失片段4构建pNC-Green-LUC
Deletion fragment 4 construct pNC-Green-LUC
UBQ5-F AAGCTCGCTCTTCTCCAGTTC 内参引物
Reference primer
UBQ5-R CACACTTGCCGCAGTAATGTC 内参引物
Reference primer
CsMIXTA-F-QPCR TCCATGCTTTACTAGGCAACAG 荧光定量引物
Fluorescent quantitative primers
CsMIXTA-R-QPCR CCACCGTCTTGTTGAGAGAG 荧光定量引物
Fluorescent quantitative primers

图1

植物表达载体构建示意图 A: 携带GUS的pCAMBIA1391; B: 携带LUC的pNC-Green-LUC; Poly(A): 花椰菜花叶病毒多聚腺苷酸化信号; HygR: 潮霉素抗性基因; 35S P: 花椰菜花叶病毒35S启动子; NOS T: 胭脂碱合酶基因终止子; Rluc: 海肾荧光素酶; NC Frame: NC克隆框架; GUS: 葡糖苷酸酶; LUC: 萤火虫荧光素酶。"

图2

CsMIXTA启动子的克隆 M: DNA分子量标准DL2000; P: 全长启动子。"

图3

CsMIXTA启动子序列分析 起始密码按照ATG的“G”为+1位点, 下画线标注的“G”为转录起始位点。"

表2

CsMIXTA启动子部分顺式作用元件"

元件
Element		 序列
Sequence		 功能
Function		 数量
Number		 位置
Position (bp)
LTR CCGAAA 低温响应元件
Cis-acting element involved in low-temperature responsiveness		 1 -2110
Box 4 ATTAAT 光响应元件
Light responsive element		 4 -1937, -1873, -1789, -1300
AE-box AGAAACAA 光响应元件
Light responsive element		 1 -806
GATA-motif GATAGGG 光响应元件
Light responsive element		 1 -293
G-box CCACGTAA 光响应元件
Light responsive element		 1 -449
ABRE ACGTG 脱落酸响应元件
Cis-acting element involved in the abscisic acid responsiveness		 1 -800
MRE AACCTAA 参与光反应性的MYB结合位点
MYB binding site involved in light responsiveness		 1 -1644
circadian CAAAGATATC 昼夜节律控制响应元件
Cis-acting regulatory element involved in circadian control		 1 -1638
TATC-box TATCCCA 赤霉素响应元件
Cis-acting element involved in gibberellin-responsiveness		 1 -176
CAAT-box CAAT\CAAAT 启动子和增强子区域中常见的顺式作用元件
Common cis-acting element in promoter and enhancer regions		 15 -2180, -2137, -1996, etc.
TATA-box TATTTAAA\TATATA\TATA\
ATTATA\ATATAT\ATATAA		 转录起始-30 bp处启动子核心元件
Core promoter element around -30 of transcription start		 84 -2034, -1980,
-1940, etc.

表3

CsMIXTA启动子转录起始位点预测"

(TATA-)转录起始位点位置
(TATA-) transcription start site position		 转录起始位点得分
Transcription start site score
-92 1.9964
-487 1.9964
-1074 1.9964
-1375 1.9879
-1835 1.9954

图4

CsMIXTA启动子缺失片段克隆 M: DNA分子量标准DL2000; P: 全长启动子; P1~P5: 启动子缺失片段1~5。"

图5

CsMIXTA启动子全长及缺失片段示意图 LTR: 低温响应元件; Box 4: 光响应元件; MRE: 参与光反应性的MYB结合位点; circadian: 昼夜节律控制响应元件; AE-box: 光响应元件; ABRE: 脱落酸响应元件; G-box: 光响应元件; GATA-motif: 光响应元件; TATC-box: 赤霉素响应元件; 图中数值单位为bp。"

图6

CsMIXTA启动子全长及缺失片段作用下的烟草GUS染色 CK: 阴性对照; P: 全长启动子; P1~P5: 启动子缺失片段1~5。"

图7

CsMIXTA启动子核心区域P4作用下的LUC荧光表达 a~c: P4-LUC注射区域; d: pNC-Green-LUC空载注射区域(阴性对照)。"

图8

CsMIXTA全长启动子作用下的工业大麻GUS染色 a: 单细胞表皮毛; b: 头状无柄腺毛; c: 头状有柄腺毛。"

图9

3种胁迫处理对CsMIXTA表达模式的影响 *、**、***分别表示在0.05、0.01和0.001概率水平差异显著。"

[1] 李秋实, 孟莹, 陈士林. 药用大麻种质资源分类与研究策略. 中国中药杂志, 2019, 44: 4309-4316.
Li Q S, Meng Y, Chen S L. A new Cannabis germplasm classification system and research strategies of non-psychoactive medicinal Cannabis. China J Chin Mater Med 2019, 44: 4309-4316 (in Chinese with English abstract).
[2] Goncalves J, Rosado T, Soares S, Simao A Y, Caramelo D, Luis A, Fernandez N, Barroso M, Gallardo E, Duarte A P. Cannabis and its secondary metabolites: their use as therapeutic drugs, toxicological aspects, and analytical determination. Medicines (Basel), 2019, 6: 31.
[3] Tanney C A S, Backer R, Geitmann A, Smith D L. Cannabis glandular trichomes: a cellular metabolite factory. Front Plant Sci, 2021, 12: 721986.
[4] Backer R, Schwinghamer T, Rosenbaum P, McCarty V, Eich Bilodeau S, Lyu D, Ahmed M B, Robinson G, Lefsrud M, Wilkins O, Smith D L. Closing the yield gap for cannabis: a meta-analysis of factors determining cannabis yield. Front Plant Sci, 2019, 10: 495.
doi: 10.3389/fpls.2019.00495 pmid: 31068957
[5] Burgel L, Hartung J, Schibano D, Graeff-Honninger S. Impact of different phytohormones on morphology, yield and cannabinoid content of Cannabis sativa L. Plants (Basel), 2020, 9: 725.
[6] Chezem W R, Clay N K. Regulation of plant secondary metabolism and associated specialized cell development by MYBs and bHLHs. Phytochemistry, 2016, 131: 26-43.
doi: S0031-9422(16)30158-3 pmid: 27569707
[7] Brockington S F, Alvarez-Fernandez R, Landis J B, Alcorn K, Walker R H, Thomas M M, Hileman L C, Glover B J. Evolutionary analysis of the MIXTA gene family highlights potential targets for the study of cellular differentiation. Mol Biol Evol, 2013, 30: 526-540.
doi: 10.1093/molbev/mss260 pmid: 23188591
[8] Matias-Hernandez L, Jiang W, Yang K, Tang K, Brodelius P E, Pelaz S.AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development in Artemisia annua and Arabidopsis thaliana. Plant J, 2017, 90: 520-534.
[9] Shi P, Fu X, Shen Q, Liu M, Tang K.The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua. New Phytol, 2017, 217: 261-276.
[10] Ewas M, Gao Y, Wang S, Liu X, Zhang H, Nishawy E M E, Ali F, Shahzad R, Ziaf K, Subthain H. Manipulation of SlMXl for enhanced carotenoids accumulation and drought resistance in tomato. Sci Bull, 2016, 61: 1413-1418.
[11] Chalvin C, Drevensek S, Dron M, Bendahmane A, Boualem A. Genetic control of glandular trichome development. Trends Plant Sci, 2020, 25: 477-487.
doi: S1360-1385(19)30350-4 pmid: 31983619
[12] Haiden S R, Apicella P V, Ma Y, Berkowitz G A. Overexpression of CsMIXTA, a transcription factor from Cannabis sativa, increases glandular trichome density in tobacco leaves. Plants (Basel), 2022, 11: 1519.
[13] Alter H, Peer R, Dombrovsky A, Flaishman M, Spitzer-Rimon B. Tobacco rattle virus as a tool for rapid reverse-genetics screens and analysis of gene function in Cannabis sativa L. Plants (Basel), 2022, 11: 327.
[14] Huang X, Chen W, Zhao Y, Chen J, Ouyang Y, Li M, Gu Y, Wu Q, Cai S, Guo F, Zhu P, Ao D, You S, Vasseur L, Liu Y. Deep learning-based quantification and transcriptomic profiling reveal a methyl jasmonate-mediated glandular trichome formation pathway in Cannabis sativa. Plant J 2024, 118: 1155-1173.
[15] Ma G, Zelman A K, Apicella P V, Berkowitz G. Genome-wide identification and expression analysis of homeodomain leucine zipper subfamily IV (HD-ZIP IV) gene family in Cannabis sativa L. Plants (Basel), 2022, 11: 1307.
[16] 张春晓, 王文棋, 蒋湘宁, 陈雪梅. 植物基因启动子研究进展. 遗传学报, 2004, 31: 1455-1464.
Zhang C X, Wang W Q, Jiang X N, Chen X M. Review on plant gene promoters. J Genet Genomics, 2004, 31: 1455-1464 (in Chinese with English abstract).
[17] 马倩, 马宝月, 穆波, 马慧. 植物基因启动子的克隆及分析的研究进展. 中国农业文摘-农业工程, 2018, 30(3): 23-29.
Ma Q, Ma B Y, Mu B, Ma H. Research progress on cloning and analysis of plant gene promoter. Agric Sci Eng China, 2018, 30(3): 23-29 (in Chinese with English abstract).
[18] 毛燕, 郑名敏, 牟成香, 谢吴兵, 唐琦. 渗透胁迫下玉米自然反义转录本cis-NATZmNAC48启动子的功能分析. 作物学报, 2024, 50: 354-362.
doi: 10.3724/SP.J.1006.2024.33013
Mao Y, Zheng M M, Mu C X, Xie W B, Tang Q. Function analysis of the promoter of natural antisense transcript cis-NATZmNAC48 in maize under osmotic stress. Acta Agron Sin, 2024, 50: 354-362 (in Chinese with English abstract).
[19] Ambawat S, Sharma P, Yadav N R, Yadav R C. MYB transcription factor genes as regulators for plant responses: an overview. Physiol Mol Biol Plants, 2013, 19: 307-321.
[20] Du H, Feng B R, Yang S S, Huang Y B, Tang Y X. The R2R3-MYB transcription factor gene family in maize. PLoS One, 2012, 7: e37463.
[21] 周悦, 赵志华, 张宏宁, 孔佑宾. 大豆紫色酸性磷酸酶基因GmPAP14启动子克隆与功能分析. 作物学报, 2022, 48: 590-596.
doi: 10.3724/SP.J.1006.2022.14016
Zhou Y, Zhao Z H, Zhang H N, Kong Y B. Cloning and functional analysis of the promoter of purple acid phosphatase gene GmPAP14 in soybean. Acta Agron Sin, 2022, 48: 590-596 (in Chinese with English abstract).
[22] Shang-Guan X X, Xu B, Yu Z X, Wang L J, Chen X Y. Promoter of a cotton fibre MYB gene functional in trichomes of Arabidopsis and glandular trichomes of tobacco. J Exp Bot, 2008, 59: 3533-3542.
[23] Livingston S J, Quilichini T D, Booth J K, Wong D C J, Rensing K H, Laflamme-Yonkman J, Castellarin S D, Bohlmann J, Page J E, Samuels A L. Cannabis glandular trichomes alter morphology and metabolite content during flower maturation. Plant J, 2020, 101: 37-56.
doi: 10.1111/tpj.14516
[24] Maes L, Inzé D, Goossens A. Functional specialization of the TRANSPARENT TESTA GLABRA1 network allows differential hormonal control of laminal and marginal trichome initiation in Arabidopsis rosette leaves. Plant Physiol, 2008, 148: 1453-1464.
[25] Xia X C, Hu Q Q, Li W, Chen Y, Han L H, Tao M, Wu W Y, Li X B, Huang G Q. Cotton (Gossypium hirsutum) JA23 and SLR1 function in jasmonate and gibberellin mediated epidermal cell differentiation and elongation. Plant Cell Tissue Organ Cult (PCTOC), 2018, 133: 249-262.
[26] Zhu L, Guan Y, Liu Y, Zhang Z, Jaffar M A, Song A, Chen S, Jiang J, Chen F. Regulation of flowering time in Chrysanthemum by the R2R3 MYB transcription factor CmMYB2 is associated with changes in gibberellin metabolism. Hortic Res, 2020, 7: 96.
[27] 赵悦, 赵艳艳, 李强锋, 段红俊. 青海茄参MYB转录因子家族生物信息学及应答低温胁迫分析. 分子植物育种, 网络首发[2023-12-06], https://link.cnki.net/urlid/46.1068.s.20231205.1916.027.
Zhao Y, Zhao Y Y, Li Q F, Duan H J. Bioinformatics analysis of MYB transcription factor family in mandragora chinghaiensis under low temperature induction. Mol Plant Breed, Published online [2023-12-06], https://link.cnki.net/urlid/46.1068.s.20231205.1916.027 (in Chinese with English abstract).
[28] Yin X, Cui Y, Wang M, Xia X. Overexpression of a novel MYB-related transcription factor, OsMYBR1, confers improved drought tolerance and decreased ABA sensitivity in rice. Biochem Biophys Res Commun, 2017, 490: 1355-1361.
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