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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (11): 2754-2763.doi: 10.3724/SP.J.1006.2024.44001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2024-11-12 Published: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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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

Table 1

Primers used in this study"

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

Fig. 1

Schematic diagram of plant expression vector construction A: pCAMBIA1391 carrying GUS; B: pNC-Green-LUC carrying LUC; Poly(A): polyadenylation signal from the cauliflower mosaic virus; HygR: hygromycin resistance gene; 35S P: cauliflower mosaic virus 35S promoter; NOS T: nopaline synthase terminator; Rluc: renilla luciferases; NC Frame: nimble cloning frame; GUS: glucosidase; LUC: firefly luciferases."

Fig. 2

Cloning the promoter of CsMIXTA M: DL2000 marker; P: full-length promoter."

Fig. 3

Sequence analysis of CsMIXTA promoter The start codon follows the ATG “G”as the +1 site and the underlined standard “G” as the transcription start site."

Table 2

Partial cis-acting elements of CsMIXTA promoter"

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.

Table 3

Prediction of the transcription start site of the CsMIXTA promoter"

(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

Fig. 4

Cloning of deletion fragment of CsMIXTA promoter M: DL2000 marker; P: full-length promoter; P1-P5: promoter deletion fragments 1-5."

Fig. 5

Schematic diagram of CsMIXTA promoter full-length and missing fragments LTR: low temperature response element; Box 4: light response element; MRE: MYB binding site involved in light responsiveness; circadian: cis-acting regulatory element involved in circadian control; AE-box: light response element; ABRE: abscisic acid response element; G-box: light response element; GATA-motif: light response element; TATC-box: erythromycin response element; TATC-box: gibberellin-responsive element; values in the figure are bp."

Fig. 6

GUS staining of transgenic tobacco with full-length and deletion fragments of the CsMIXTA promoter CK: negative control; P: full-length promoter; P1-P5: promoter deletion fragments 1-5."

Fig. 7

Fluorescence expression of LUC in tobacco under P4, the core region of CsMIXTA promoter a-c: P4-LUC injection area; d: pNC-Green-LUC injection area (negative control)."

Fig. 8

GUS staining of transient transgenic industrial hemp under the action of a full-length promoter a: unicellular trichomes; b: capitate-sessile glandular trichomes; c: capitate-stalked glandular trichomes."

Fig. 9

Effects of three stresses on the expression activity of CsMIXTA *, **, and *** indicate significant difference at the 0.05, 0.01, and 0.001 probability levels, respectively."

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