Establishment of an Efficient Transient Transformation System for Tagetes erecta Corollas and Analysis on the Promoter Activity of TeCYC2c Gene

doi:10.11983/CBB24150

Abstract

Abstract: INTRODUCTION: Marigold (Tagetes erecta), an important ornamental and medicinal plant, has a unique capitulum characteristic of the Asteraceae family with distinct ray and disc florets. However, the lack of efficient genetic transformation system has limited the research on the mechanism of floral organ development of marigold. Floral transient transformation system offers a rapid approach to study the function of genes expressed specifically in floral organs. This study aimed to establish an efficient transient transformation system for marigold corollas and to analyze the promoter activity of TeCYC2c, which highly expressed in corollas, thereby laying the technical foundation for the rapid verification of floral gene function.
RATIONALE: A fusion expression vector, incorporating the CaMV35S promoter and the GUS reporter gene, was constructed to facilitate the transient transformation in marigold corollas. The study delved into the effects of bacterial strain type (GV3101, LBA4404, EHA105), bacterial suspension concentration (OD₆₀₀ values 0.5-2.0), infection duration (10- 40 min), and co-culture time (1-4 d) on the transient transformation efficiency of the GUS gene. Based on this transient transformation system for the marigold corollas, the promoter activity of the TeCYC2c gene was investigated. A 1 735 bp upstream sequence of the TeCYC2c gene was cloned and four promoter deletion expression vectors, with the GUS gene as the reporter gene, were constructed based on the distribution and quantity of elements predicted by PlantCARE. Subsequently, these vectors were employed for transient transformation of marigold corollas to facilitate an in-depth analysis of promoter activity.
RESULTS: The transient transformation efficiency in marigold corollas demonstrated that the GV3101 strain achieved the highest infection efficiency; the bacterial suspension concentration, quantified at an OD₆₀₀ value of 1.0, yielded the most robust transformation efficiency; the infection time was observed to exert no substantial influence on transient transformation efficacy; moreover, a co-culture time of 24 hours was identified as the optimal condition for the process. The results of GUS staining and GUS activity assay revealed that the core region of the promoter was located at -650 to -1 bp. It was speculated that the light-responsive elements within this region positively up-regulated the expression of downstream genes, while the hormone-responsive and stress-responsive elements unique to pTeCYC2c (-1 735) and pTeCYC2c (-1 406) might have an inhibitory effect on promoter-driven downstream gene expression.
CONCLUSION: This study established an efficient transient transformation system for marigold corollas, optimized through strain selection and parameter tuning. The identification of the TeCYC2c promoter core region (-650 to -1 bp) and its regulatory elements provides critical insights into the regulatory mechanism of the TeCYC2c gene. The transient transformation system and promoter analysis method lay a technical foundation for accelerating functional studies of floral development genes in marigold, with potential applications in the genetic improvement of ornamental plants.

GUS staining of marigold (Tagetes erecta) corollas under different transient transformation conditions. (A) GUS staining of marigold corollas infected by three different bacterial strains; (B) GUS staining under four different concentrations (OD₆₀₀) of bacterial suspension; (C) GUS staining under four different infection duration; (D) GUS staining under four different co-culture time. (A)-(D) Bars=1 cm

Key words: marigold (Tagetes erecta), TeCYC2c, promoter, transient transformation, GUS activity

Linlin Dou, Yu Zhu, Cuicui Liu, Yunping Zang, Zhengguo Tao, Manzhu Bao, Yanhong He. Establishment of an Efficient Transient Transformation System for Tagetes erecta Corollas and Analysis on the Promoter Activity of TeCYC2c Gene[J]. Chinese Bulletin of Botany, 2025, 60(6): 875-887.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB24150

https://www.chinbullbotany.com/EN/Y2025/V60/I6/875

Figures/Tables 6

Table 1 Primer sequences

Primer name	Sequences (5′→3′)	Function
pTeCYC2c-F (-1735)	ACGACAATGAAGTTTGAAGACACAG	Promoter cloning
pTeCYC2c-F (-1406)	ATTTGAAGTTGCAACTCTGTCATAAAT	Promoter deletion fragments cloning
pTeCYC2c-F (-1000)	AACCATTAGGGCCGTGTCG	Promoter deletion fragments cloning
pTeCYC2c-F (-650)	GGTGTCTTGGTAATTTTAAAAAAACG	Promoter deletion fragments cloning
pTeCYC2c-R	TGTATTTTGGAATTGAAATGTGAAATAA	Promoter deletion fragments cloning

Figure 1 GUS staining of marigold (Tagetes erecta) corollas under different transient transformation conditions (A) GUS staining of marigold corollas infected by three different bacterial strains; (B) GUS staining under four different concentrations (OD600) of bacterial suspension; (C) GUS staining under four different infection duration; (D) GUS staining under four different co-culture time. (A)-(D) Bars=1 cm

Figure 2 The influence of strain type (A), bacterial suspension concentration (B), infection time (C), and co-culture time (D) on the transient transformation efficiency of Tagetes erecta corollas Different lowercase letters on the histogram indicate significant differences at the P<0.05 level.

Table 2 Analysis of the cis-acting elements in the TeCYC2c promoter

Cis-regulatory elements	Sequences and position	Number	Description
ARE	AAACCA: -1359, -1296	2	Cis-acting regulatory element essential for the anaerobic induction
LAMP-element	CTTTATCA: -1661	1	Part of a light responsive element
MBS	CAACTG: -1712	1	MYB binding site involved in drought-inducibility
TCT-motif	TCTTAC: -603	1	Part of a light responsive element
Box 4	ATTAAT: -1420, -429, -413, -409	4	Part of a conserved DNA module involved in light responsiveness
ACE	CTAACGTATT: -630	1	Cis-acting element involved in light responsiveness
CGTCA-motif	CGTCA: -1690, -1347, -1235, -797, -713	5	Cis-acting regulatory element involved in the MeJA- responsiveness
MBSI	aaaAaaC(G/C)GTTA: -633	1	MYB binding site involved in flavonoid biosynthetic genes regulation
GT1-motif	GGTTAA: -1002, -67	2	Light responsive element
ABRE	ACGTG: -1349	1	Cis-acting element involved in the abscisic acid responsiveness
TATC-box	TATCCCA: -1708	1	Cis-acting element involved in gibberellin-responsiveness
G-box	CACGAC: -1655, -1504	2	Cis-acting regulatory element involved in light responsiveness
O²-site	GATGATGTGG: -981	1	Cis-acting regulatory element involved in zein metabolism regulation
AE-box	AGAAACAA: -74	1	Part of a module for light response
LTR	CCGAAA: -1496	1	Cis-acting element involved in low-temperature responsiveness

Figure 3 Schematic diagram of the design of TeCYC2c gene promoter deletion fragments and cloning of different length fragments of the promoter (A) Schematic diagram of the design of TeCYC2c gene promoter deletion fragments (Box 4: Light-responsive element; ACE: Light-responsive element; TCT-motif: Light-responsive element; GT1-motif: Light-responsive element; AE-box: Light-responsive element; ARE: Anaerobic induction element; MBS: Drought induction element; CGTCA-motif: MeJA-responsive element; ABRE: ABA-responsive element; TATC-box: GA-responsive element; LTR: Low-temperature responsive element; the numerical values in the figure are in bp); (B) Cloning of different length fragments of the TeCYC2c gene promoter (M: DL2000 marker; 1-4: Representing pTeCYC2c (-1 735), pTeCYC2c (-1 406), pTeCYC2c (-1 000), and pTeCYC2c (-650), respectively).

Figure 4 GUS staining and GUS activity assay driven by the promoters with different lengths (A) Negative control; (B) GUS staining under the influence of pTeCYC2c (-1 735); (C) GUS staining under the influence of pTeCYC2c (-1 406); (D) GUS staining under the influence of pTeCYC2c (-1 000); (E) GUS staining under the influence of pTeCYC2c (-650); (F) Determination of GUS activity driven by the promoters with different length. Different lowercase letters on the bar graph indicate significant differences at the P<0.05 level. (A)-(E) Bars=1 cm

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