In this study, we investigated the application of mCherry, a fluorescent protein, to evaluate the inducible promoter rd29A in Arabidopsis under low temperature, using the Arabidopsis genome as a template to amplify this promoter by PCR. Overlapping extension PCR was used to ligate the rd29A promoter and the mCherry gene to initiate the expression of mCherry through construction of a pHDE-rd29A-mCherry plasmid. Competent Agrobacterium cells were transformed with the recombinant plasmid, and Agrobacterium-mediated transfect Arabidopsis to obtain the progeny which was identified containing rd29A-mCherry in the transformed plants. Positive transgenic T1 seeds were planted in 1/2MS medium, and the seedlings were subsequently cultured at 34, 24, 14, 4 and 0° C for 20 h. Expression of the mCherry gene was then determined using an inverted fluorescence microscope. The results showed that fluorescence intensity was significantly higher in seedlings grown at 4° C compared with those grown at other temperatures. Furthermore, the rd29A promoter was induced to express low-levels of mCherry expression. Quantitative PCR assay indicated that mCherry expression was high under the 4° C condition, which was consistent with the results obtained by microscopy. These findings indicate that mCherry can be used as an exogenous marker to investigate spatio-temporal promoter activation.

• rd29a Promoter

• Overlapping Extension PCR Technology

• mCherry Gene

• Marker Gene

Su M, Wang Z, Ouyang L, Li L (2024) Application of the Fluorescent Protein mCherry to Study the Inducible Promoter rd29a under Low Temperature Conditions in Arabidopsis. Int J Plant Biol Res 12(1): 1140.

Gene promoters are key elements that regulate gene expression, and exist in constitutive, tissue-specific, and inducible forms [1]. The Arabidopsis rd29A promoter is induced in response to adverse conditions [2], and comprises one Abscisic Acid (ABA)-Responsive Cis-Acting Element (ABRE) sequence, two DNA Replication-Related Elements (DREs), and two DRE- related CCGAC sequences [3]. The total length of the promoter is 1446 bp; however, responses to drought, high salinity, and low temperature stress are regulated by the region between base pairs -174 to -55 [4]. rd29A, also known as LTI78 and COR78, carries three introns at the same position as those in rd29B [5]. Evidence suggests that retention of the DRE element within the rd29A promoter enables rapid induction of the rd29A gene under conditions of high salinity, drought, or low temperature stress, indicating that the rapid expression of this gene does not depend on the action of intracellular ABA. In addition to Arabidopsis, the inducible rd29A promoter has been reported in tobacco and wheat [6]. Furthermore, the GUS reporter gene has been experimentally introduced into potatoes, and GUS activity has been detected in transgenic potato plants under stress conditions [7]. Research using transgenic plants has commonly utilized the constitutive 35S promoter; however, the constitutive expression of downstream genes often leads to slow plant growth [1]. As rd29A is a stress-inducible promoter, the expression of downstream genes is only increased in the presence of external stress. Thus, when the stress is removed, gene expression will cease which preserve the plant’s resources and does not impact growth.

Fluorescent proteins have wide applications and play important roles in biological research involving live cell imaging and analyses of protein expression. mCherry encodes a red fluorescent protein, which was originally extracted from mushroom coral, and can be used as a marker to track target molecules and cellular components. Compared with Green Fluorescent Protein (GFP), mCherry has longer excitation and emission wavelengths and is not affected by the chlorophyll auto fluorescence of green tissues [8]. Therefore, it can also be used with GFP without the risk of cross-reaction. Transgene- free progeny were easily obtained via mCherry fluorescence screening in our previous study. In this study, we investigated the application of mCherry, a fluorescent protein, to evaluate the inducible promoter rd29A in Arabidopsis under low temperature. The use of mCherry as a reporter gene can aid identification of the expression efficiency of the promoter.

Test Materials

The mCherry-containing PHDE-mCherry plasmid was obtained from Professor Zhao of the University of California (San Diego). Col-0 wild-type Arabidopsis thaliana was grown in the plant culture room of our lab.

Main Test Reagents

The plasmid small extraction kit, agarose gel DNA recovery kit, Taq DNA Polymerase (ET101), and DNA Marker were purchased from Beijing Tiangen Biochemical Technology Co. Ltd (China); kanamycin and DNA loading buffer were obtained from Shanghai Biotech Biotechnology (China). Epicentre T5 exonuclease, NEB Taq DNA Ligase, Bsa1 restriction enzyme, and NEB Phusion DNA polymerase were purchased from New England Biolabs (USA), and Dithiothreitol (DTT) was purchased from Beijing Biyuntian Biotechnology (China).

Primer Design

Primers were designed using the   Primers   5   software and synthesized by Shanghai Bio-Engineering and Biological Engineering Co, Ltd (Table 1).

Table 1: Primer sequences.

Construction of the PHDE-rd29a-mCherry Expression Vector

Amplification of rd29a And mCherry: Using wild-type Arabidopsis genomic DNA as a template, PCR amplification was performed using rd29A-F and rd29A-R primers (Table 1). The reaction was performed as follows: pre-denaturation at 94°C for 4 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension 72°C 45 s; final extension at 72°C for 2 min; and storage at 4°C. The PCR product was analyzed by 1% agarose gel electrophoresis, and the target band was excised and recovered using an agarose gel DNA extraction kit. (Tiangen Biochemistry Company, Beijing).

The pHEE401E-mCherry plasmid was used as a template, and mCherry-F and mCherry-R were used as primers for PCR amplification (Table 1). The reaction was performed as follows: pre-denaturation at 94°C for 4 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s; final extension at 72°C for 8 min; and storage at 4°C. The PCR product was analyzed by 1% agarose gel electrophoresis, and the target band was excised and recovered using an agarose gel DNA extraction kit (Tiangen Biochemistry Company, Beijing).

rd29a-mCherry Fusion PCR Amplification: With reference to overlapping fusion PCR technology [9-10], PCR was performed using the rd29A promoter with the mCherry sequence as a template after amplification and purification, and rd29A-F and mCherry-R as primers. Denaturation was performed at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s; final extension at 72°C for 8 min; and storage at 4°C. The PCR product was analyzed by 1% agarose gel electrophoresis, and the target band was excised and recovered using an agarose gel extraction kit (DNA gel extraction kit from Tiangen Biochemistry Company, Beijing).

Digestion and Homologous Recombination of Plasmid PHDE-mCherry: The PHDE-mCherry plasmid was digested with the restriction enzyme Bas1. The reaction was performed in a 20 µL system at 37°C for 16 h. The undigested plasmid was used as a control. The digested product was verified by 1% agarose gel electrophoresis.

After purification and recovery of rd29A-mCherry, the target fragment and digested product were subjected to homologous recombination in a 4.3 µL reaction system (recombinant enzyme 3 µL, digested plasmid 0.3 µL, target fragment 1 µ) and a water bath at 50°C for 1 h.

Recombinant Products PCR Analysis of Bacterial Solution: Recombinant products were introduced into DH5α competent cells using the heat-shock method [11]. According to the manufacturer protocol provided by Guangzhou Dingguo Biotechnology Company (Beijing), the transformed DH5α competent cells were plated on LB solid medium and cultured for 12 h. Single colonies were then selected and identified by PCR using the following reaction procedure: pre-denaturation at 94°C for 4 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s; extension at 72°C for 8 min; and storage at 4°C.

Recombinant Plasmid PHDE-rd29a-mCherry Identification: PCR-positive bacterial culture was cultured in LB liquid culture medium (containing 50 mg/mL kanamycin at a volume ratio of kanamycin: LB liquid culture medium of 1:1000). The bacteria were shaken at 37°C for 12 h, and the recombinant plasmid was extracted using the plasmid small extraction kit, and subsequently analyzed via PCR. The reaction conditions were the same as those described above for bacterial solution.

The positive recombinant plasmids were selected and sent to Sheng Gong Bioengineering. (Guangzhou, China) for sequencing, and sequence comparisons were verified using DNAMAN software.

Transformation of Arabidopsis

Transformation of Recombinant Plasmid into Agrobacterium and Preparation of Dip Solution: The successfully sequenced plasmids were transferred into Agrobacterium competent cells. Transformation was performed according to the protocol provided by Shanghai Veidi Biotechnology Co. Ltd. (Product No. AC1001). The transformed Agrobacterium GV3103 competent cells were transferred to LB solid medium and cultured at 28°C for 48 h. Single colonies were picked and identified by PCR, using the following procedure: pre- denaturation at 94°C for 4 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 45 s; a final extension at 72°C for 8 min; and storage at 4°C.

Following validation by agarose gel electrophoresis, the positive bacterial solution was selected for expansion and culture in LB liquid medium and the bacteria were shaken at 28°C for 16 h. The bacterial cells were collected and suspended in 100 mL dipping buffer (5% sucrose solution, 0.02% Silwet L-77) for dipping solution to transform Arabidopsis.

Arabidopsis Dipstick: Col-0 wild-type Arabidopsis seeds were planted and topped following the appearance of flower buds. Once flower buds appeared on the side branches, all flowering flowers and pods were excised 1 day before dipping.

The buds were dipped in Agrobacterium infusion solution and stirred gently for 1-2 min. The liquid remaining on the leaves and stems was then removed with absorbent paper. After 48 h of dark culture, Full day light culture at 23°C was performed. The dipping process was repeated following the growth of new buds.

Treatment and Identification of  Transformed Offspring

PCR Identification after Transformation: The transformed T0 generation Arabidopsis seeds were collected, and then planted to obtain T1 seedlings. The DNA of T1 leaves was extracted and used as a template. The target sequence was amplified by PCR using rd29A-F and mCherry-R primers (Table 1). The reaction was performed as follows: pre-denaturation at 94°C for 4 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s; final extension at 72°C for 2 min; and storage at 4°C. The size and identity of the target bands were verified by 1% agarose gel electrophoresis.

Low Temperature Treatment of Buds and Identification: The obtained T1 seedlings were cultured under temperatures of 34, 24, 14, 4 and 0°C for 20 h. Then, the intensity of red fluorescence in the seedlings was observed using an inverted fluorescence microscope (Leica, Germany). Excitation wavelength is 587 nm.

Determination of Temperature-Induced MCherry Expression Driven by rd29a: RNA was extracted from transgenic seedlings under different temperature treatments using the Easy Pure Plant RNA Kit (TransGen Biotech, Beijing), following the manufacturer’s instructions. CDNA was obtained by reverse transcription for subsequent use in qPCR. The reaction system was 20 μL, including 2 μL of cDNA, 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 10μL of 2× Transcript Tip Green qPCR Super Mix, 0.4 μL of passive reference dye (50×), and 7.2 μL of nuclease-free water. Each sample was analyzed in triplicate for each pair of primers, and kept on ice. Relative expression of the mCherry gene under different temperatures was calculated using the 2-ΔΔCT method.

Construction of the PHDE-rd29a-mCherry Expression Vector

Following digestion of PHDE-mCherry with Bsa1, the plasmid was linearized, and the electrophoretic mobility was found to be reduced compared with the non-digested plasmid (Figure 1A). Analysis of the 2000 bp DNA marker revealed that the amplified product No. 1 was between 500 and 250 bp; No. 2 was between 750 and 500 bp, and No. 3 was between 1000 and 750 bp. Location of the expected electrophoretic band of the mCherry sequence (263 bp), rd29A promoter (662 bp), and the splicing sequence of rd29A promoter and mCherry (925 bp), indicating the amplified product band was the correct size and had been successfully recovered and purified (Figure 1B), (DNA gel extraction kit from Tiangen Biochemistry Company, Beijing).

Figure 1: Construction of the PHDE-rd29A-mCherry expression vector

A: Enzymatically cut vector, 2: undigested vector;

B. M: DL2000 DNA marker, 1: mCherry sequence, 2: rd29A promoter, 3: rd29A promoter and full-length mCherry Sequence,

C. M: DL2000 DNA marker, 1-7. PCR analysis of culture containing bacteria transformed with recombinant plasmid.

D. M: DL2000 DNA marker, 1-3. PHDE- rd29A -mCherry- PCR results for recombinant plasmid.

The recombinant plasmid was transformed into DH5α competent cells, which were then screened on a kanamycin- resistance plate. Subsequently, a single colony was picked, cultured, and identified by PCR analysis of the E. coli culture (Figure 1C). The amplified bands were located between 1000 and 750 bp as expected (925 bp), with a 100% positive rate.

High-quality PCR bands from the bacterial culture were selected for expansion and culture. The plasmid was extracted and PCR was performed to identify the extracted plasmid (Figure 1D). The band size was consistent with expectations, indicating that construction of the PHDE-rd29A-mCherry expression vector was successful. The samples were then sent for sequencing, and the sequences were aligned (Figure 2) to confirm successful construction of the PHDE-rd29A-mCherry expression vector.

Figure 2: Sequence alignment results

Agrobacterium Transformation with PHDE-rd29a- mCherry Recombinant Plasmid

Following the transformation of recombinant plasmid into Agrobacterium GV3103 competent cells, PCR was performed d on the bacterial solution to confirm whether the transformation was successful, the results (Figure 3A) showed that the band size was between 1000 and 750 bp, which was consistent with the expected size. These results indicate that the transformation was successful.

Figure 3: PCR identification of recombinant plasmid PHDE-rd29A-mCherry and PCR analysis of rd29A-mCherry in T1 plant

A: PCR identification of recombinant plasmid PHDE-rd29A-mCherry. M: DL2000 DNA marker, 1-5: PCR results of Agrobacterium tumefaciens transformed with recombinant plasmid.

B: PCR analysis of rd29A-mCherry in T1 plant. M: DL2000 DNA marker, 1-3: PCR results of rd29A-mCherry in T1 plant

Visual Screening and Molecular Identification of Transformed Offspring

Identification of Positively Transformed Offspring: To verify whether the T1 generation plants contained the mCherry gene, DNA was extracted from the T1 leaves of transformed offspring and subjected to PCR identification (Figure 3B). The results revealed a band between 1000 and 750 bp in size, which was as expected. This confirmed that seedlings of the T1 generation contained the mCherry gene and that the transformation had been successful.

Observation of rd29a Promoter Activity Under Low Temperature Conditions: Seedlings expressing the red fluorescent protein were planted on culture medium and cultured at 34, 24, 14, 4 and 0°C for 20 h. Expression of the mCherry gene,under the control of the rd29A promoter, was observed in young shoots grown under low temperature (4°C). Furthermore, the red fluorescence was significantly higher at 4°C compared with the other temperatures analyzed. (Figure 4), this demonstrates that the rd29A promoter is activated and exerts strong activity under low temperatures (4°C).

Figure 4: Color development of young shoots following induction at low temperatures under fluorescence microscope

Figure 5: Histogram of mcherry relative expression under different temperature treatments. Note: *indicate significant difference (p = 0.05) **indicate extremely significant differences (p = 0.01)

Low-Temperature-Induced rd29a Promoter Induced: mCherry Expression Efficiency Identification By Fluorescent Quantitative PCR: Graph Pad Prism 8 was used to analyze the difference significance (T-test) of the exported Ct value data and to draw a histogram for display with the samples treated at 24°C as the control. Expression of the mCherry gene was significantly higher compared with the other treatment conditions, and the difference reached a very significant level, indicating that expression was induced under low temperature. mCherry expression was significantly higher under 4°C treatment compared with the other temperatures. As the temperature increased, mCherry gene expression gradually decreased. mCherry expression patterns were consistent with the results observed for fluorescence(Figure 5), indicating that fluorescence intensity can be used as a phenotypic marker to identify gene expression driven by the promoter.

Many studies have investigated stress-resistance genes in plants. Most studies have constructed expression vectors using constitutive promoters to drive downstream gene expression [12]. Most studies describing the genetic transformation of crops have shown that expression vectors constructed using constitutive promoters can improve the resistance of transgenic plants. This is because constitutive promoters can drive the expression of downstream genes throughout the growth and development of the plant. In addition, there is no temporal or spatial specificity for such promoters, and they are not affected by external factors. The continuous excessive production of heterologous proteins or metabolic by-products leads to excessive consumption of resources within the plant, hindering normal growth and development, potentially disrupting the balance of plant metabolism, with often-toxic side effects [4]. Tissue-specific promoters are expressed and regulated in specific tissues and organs, and they are rarely used. Therefore, studies utilizing inducible promoters that drive the expression of target genes under specific stimuli are particularly important, and are a focus of current research [12]. Plant stress refers to environmental factors that induce damage to plant cells, including abiotic stresses such as low temperature and drought, and biological stresses such as pathogens [1].

The rd29A promoter has been widely studied as an example of an inducible promoter, and has been used in many breeding studies to improve the resistance of crops [4,7]. The rd29A promoter contains the DRE core sequence and the ABRE [13]. When the plant is exposed to low temperature, ABA signaling in the plant interacts with the ABRE sequence of the rd29A promoter and subsequently initiates the expression of rd29A.

Fluorescent proteins have a wide range of applications and play important roles in biological research [14]. mCherry encodes a red fluorescent protein, which is often fused to specific promoters. To study the activity of specific promoters [15-17]. Fused mCherry with specific promoters to screen and identify positively transformed plants. The expression vector containing the stress-inducible promoter rd29A and the mCherry fusion gene use low temperature to induce the rd29A promoter to enhance the expression of downstream genes. In addition, because mCherry is a reporter gene that can be monitored by red fluorescence, the strength and extent of promoter activity in transgenic plants can be directly observed under a fluorescent microscope. The combination of these approaches greatly enhances research into promoters, and provides information for studying the spatio- temporal activity of the promoter.

This research was supported by The National Natural Science Foundation of China (32071780); the Science and Technology Tackle Key Problem of Guangdong Province (2021S0074, 2022DZXHT072, 2023S018087).

Lejun Ouyang and Limei Li conceived and designed the experiments; Min Su, Zechen Wang, Chaohong Wang, performed the experiments and analyzed the data; Min Su, Zechen Wang wrote the paper.

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