Function Deletion Analysis of Light-induced Gacab Promoter from Gossypium arboreum in Transgenic Tobacco

ACTA AGRONOMICA SINICA Volume 35, Issue 6, June 2009 Online English edition of the Chinese language journal Cite this article as: Acta Agron Sin, 2009, 35(6): 1006–1012.

RESEARCH PAPER

Function Deletion Analysis of Light-induced Gacab Promoter from Gossypium arboreum in Transgenic Tobacco WANG Xu-Jing, LI Wei-Min, TANG Qiao-Ling, JIA Shi-Rong, and WANG Zhi-Xing* Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Abstract: The 1009 bp promoter sequence of a light-induced gene, cab, has been cloned from the Gossypium arboreum in an earlier study. This gene encodes the chlorophyll a/b binding protein, which belongs to a class of light-inducible proteins. For designing a minimal length light-inducible Gacab promoter, the full-length Gacab P and the 5’ truncations of 197, 504, and 779 bp in length were fused with gus (uid A) gene and ligated into plant expression vectors. All the constructs were transformed into Nicotiana tabacum variety NC89 using Agrobacterium-mediated transformation method. Seeds from the T1 generation of transgenic tobacco were germinated and grown in either light or dark condition. A GUS histochemical assay showed that only the full-length Gacab promoter was light inducible and expressed in a tissue-specific manner. Promoter activities were quantified by GUS quantity analysis, and the promoter fragment from −504 to −1 bp had the highest activity, which was 0.6-fold higher than that of the CaMV35S promoter. Keywords: Gacab promoter; function deletion analysis; transgenic tobacco

A large array of structural, metabolic, and regulatory genes are coordinately expressed during plant photomorphogenesis. Among them, the light-inducible genes are those gene highly expressed in light-grown seedlings but poorly expressed in dark-grown ones. The expression level of light-inducible genes is diversified in response to variation in light quality, intensity, direction, and photoperiod. Some of these genes are tissue-specific and highly expressed in leaves but not in roots. Genes encoding plant chlorophyll a/b binding proteins (CAB) are a typical group of light-inducible genes. cab genes have been isolated from a variety of plant species including rice (Oryza sativa L.) [1], pea (Pisum sativum L.) [2], petunia (Petunia hybrida Vilm.) [3], Lemna gibba [4], wheat (Triticum aestivum L.) [5], and Arabidopsis thaliana [6]. It is well established that cab promoters in plants are light- inducible and tissue-specific [7]. Expression of cab genes is controlled by different photoreceptors and regulated primarily at the level of transcription [8]. The expression regulatory elements of cab genes are localized in the 5’ noncoding region. Notably, a 400 bp 5’

upstream region of pea cab is sufficient for both organ specification and light regulation in transgenic tobacco (Nicotiana tabacum) [9]. A 5’ upstream region of A. thaliana cab1 1396 bp in length contains at least 3 cis-acting elements that are involved in the light-dependent developmental expression of cab1 gene. Among these elements, one is essential for promoter specificity, which is located between −253 bp and −158 bp; another one, located between −321 bp and −253 bp, appears to be a modulator that enhances the cab promoter activity. Sequence analysis suggests that a potential Z-DNA- forming sequence (ATACGTGT) is involved in lightdependent developmental expression of the cab-1 gene [10]. A 268 bp fragment of the wheat cab-1 promoter functions as a light-responsive and organ-specific enhancer. This fragment contains at least 3 distinct elements that contribute to leafspecific expression in transgenic tobacco [7]. Certain cis elements in the cab promoter have been identified and characterized [11–14]. The Gacab promoter has been cloned from Gossypium arboreum. The full-length promoter is 1009 bp [15]. According

Received: 10 October 2008; Accepted: 17 February 2009. * Corresponding author. E-mail: [email protected] Copyright © 2009, Crop Science Society of China and Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Published by Elsevier BV. All rights reserved. Chinese edition available online at http://www.chinacrops.org/zwxb/ DOI: 10.1016/S1875-2780(08)60086-3

WANG Xu-Jing et al. / Acta Agronomica Sinica, 2009, 35(6): 1006–1012

to the PlantCARE analysis results of Gacab promoter sequence and the location of light regulation elements, we designed a series of 5’ deletions of this promoter. To design a minimal length light-inducible Gacab promoter, we used gus expression in tobacco plants monitored by fluorescence as an activity reporter for a series of 5’ deletions of this promoter. These constructs were created using an Agrobacteriummediated transformation method. The results indicate that only the full-length promoter is light-inducible. GUS quantitative analysis results indicate that the promoter fragment from 504 to –1 bp has the highest activity, which was 0.6-fold higher than that of the CaMV35S promoter. The CaMV35S promoter has a nature of constitutive expression and is used usually to drive gene expression in genomic modified plants.

1 1.1

Materials and methods Materials

Nicotiana tabacum variety NC89, transgenic tobacco with pBI121, Escherichia coli DH5Į, Agrobacterium tumefaciens strain LBA4404, and vector pBI121 were maintained in our laboratory. Restriction enzymes, Taq enzyme, and T4 ligase were purchased from the TaKaRa (Dalian, China). All other chemicals were in analytical grade. 1.2 Construction of plant expression vectors harboring different lengths of Gacab promoter PCR was conducted to amplify different lengths of the Gacab promoter. The sequences of the primers along with the underlined restriction enzyme sites are as follows: p-A (5’-GC AAGCTTGTCTAATAAATAATG-3’), p-B (5’-CAAGCTT AGACCAACACCACTGACT-3’), p-C (5’-CTAAGCTTCA ACATCAAGGGAGTTGT-3’), p-D 5’-CTAAGCTTGTGGA TTAAAGATTGCC-3’), and p-R (5’-CTGGATCCGGTTGT AGAGGCCATTGTG-3’). Using the Gacab promoter as a template, DNA amplification was performed using the paired primers p-A/p-R, p-B/p-R, p-C/p-R, and p-D/p-R to generate fragments A (1009 bp), B (779 bp), C (504 bp), and D (197 bp), respectively. PCR conditions were 96°C for 3 min; 94°C for 30 s, 50°C for 1 min, and 72°C for 1 min for 30 cycles; and a final extension at 72°C for 10 min. The products amplified were digested with Hind III and BamH I and inserted into pBI121 (with kanamycin resistance) to make plant expression vectors pA, pB, pC, and pD, in which the CaMV35S promoter was replaced by Gacab promoters. These expression vectors were able to express in all organs and throughout the whole growing period of a plant. 1.3

Transformation of tobacco plants

Vectors pA, pB, pC, and pD were transformed into A. tumefaciens strain LBA4404 using the electroporation method.

LBA4404 was grown on YEB medium at 28°C and shaken at 150–250 r min−1 overnight. Cultures were diluted 1:1 with YEB and allowed to grow to A550 of 1.0. For cocultivation experiments, leaf sections from 3- to 4-week-old tobacco shoot cultures were used. After the leaf sections were treated with A. tumefaciens, they were placed on a cocultivation medium (MS salts/ 3% sucrose) and incubated at 28°C in the dark for 4 d. Leaf sections were selected on medium containing MS salts, 3% sucrose, 2 mg L−1 6-benzylaminopurine, 500 mg L−1 carbenicillin, and 100 mg L−1 kanamycin. 1.4

Histochemical GUS assay

GUS assay was performed as described by Jefferson et al. [16]. Plant tissues or leaf sections were soaked into X-gluc solution sealed closely to avoid evaporation, incubated overnight at 37°C in the dark, and fixed in formalin-isopropyl alcoholglacial acetic acid (FAA) solution for 15 min. To remove chlorophyll thoroughly, the fixed leaf sections were rinsed successively in 75%, 85%, 95%, and 100% ethanol. 1.5

GUS quantity analysis

Fresh leaves of 100 mg from a transgenic plant were ground in liquid nitrogen with a mortar and a pestle, and mixted with 600 μL cold GUS extraction buffer, containing 0.05 mol L−1 Na3PO4 (pH 7.0), 0.01 mol L−1 β-mercaptoethanol, 0.01 mol L−1 ethylene diamine tetraacetic acid (EDTA), 0.1% (W/V) sarcosyl, and 0.1% (W/V) Triton X-100 until homogenized. The sample was centrifuged at 4000×g for 15 min. The supernatant was used to measure the protein concentration according to the method described by Bradford [17]. Standard dilutions of 4-methylumbelliferone (4-MU) in carbonate stop buffer were prepared. Each dilution (300 μL) in duplicate was placed into a microtiter plate. The fluorescence was measured with an excitation of 365 nm and an emission of 455 nm using a Kontron SFM 25 spectrofluorimeter (Switzerland), and the slit widths were set at 10 nm. The calibration curve of 4-MU was drawn according the fluorescence data. To quantify the level of GUS activity, 10 μL of plant extract was added to 500 μL GUS assay buffer [1 mmol L−1 4-methylumbelliferyl-ȕ-D-glucuronide (MUG) in GUS extraction buffer]. The reaction was incubated at 37°C for 30 min and terminated by adding carbonate stop buffer (0.2 μmol L−1 Na2CO3) to a final volume of 1.5 mL. The fluorescence of each sample was measured (excitation 365 nm and emission 455 nm) using a Kontron SFM 25 spectrofluorimeter with the slit widths of 10 nm. Fluorescence units were converted to picomoles (pmol) of 4-MU using a calibration curve as described above. GUS activity was expressed as pmol 4-MU formed per minute per mg protein according to the equation G = 1510 × (e/30) × d, where G is the activity of GUS protein, e is the concentration of 4-MU, and d is the concentration of

WANG Xu-Jing et al. / Acta Agronomica Sinica, 2009, 35(6): 1006–1012

soluble protein. The experiment was repeated for 3 times.

light-induced and organ-specific expression patterns.

1.6 Expression pattern of Gacab promoter and its 5’-deletion mutants

2

Seeds of the T1 transgenic tobacco with different lengths of the Gacab promoter were geminated in the dark, and the seedlings were then cultured in light for 2 d. Simultaneously, another group of T1 seeds were germinated in the light. Histochemical GUS assays of all transgenic tobacco growing at both dark and light conditions were performed to detect the

Gacab promoter

CAAT

−1009

Results

2.1 Plant expression vectors with different lengths of Gacab promoter expressed in transgenic tobacco A series of 5’-end-deleted Gacab promoter fragments were amplified and cloned into the pBI121 plant expression vector. The resulting plasmids contained the defined promoter sequences A through D (Fig. 1). The constructs were analyzed ATG

gus

T nos pA

−779

pB −504

pC −197

pD pBI121

35S promoter

Fig. 1 Construction of plant expression vectors containing Gacab promoter 5’ deletion mutant A series of 5’-end-deleted Gacab promoter fragments were amplified using PCR method. Amplified products were digested with Hind III and BamH I and inserted into pBI121 to make plant expression vectors pA, pB, pC, and pD, in which the CaMV35S promoter was replaced by Gacab promoters. The coordinates of the relative deletion fragment are indicated. The relative positions of the CAAT box and the transcription starts site (ATG) are shown.

after transformation into transgenic plants. A total of 30 to 35 independent transgenic tobacco lines were generated with each of these constructs. Histochemical GUS assay results demonstrated that the gus gene, driven by the Gacab promoter fragments, was expressed effectively to produce detectable proteins (Fig. 2). 2.2 Analysis of expression of Gacab promoter constructs in transgenic plants To analyze the effectiveness of different lengths of the Gacab promoter, we measured GUS activity in transgenic tobacco plants. GUS expression under the control of the Gacab promoter deletion constructs was examined by fluorometric assays using leaf protein extracts. Twenty independent lines for each construct were selected to monitor their GUS activities. The average GUS activity is presented for each construct (Fig. 3). The GUS activity increased when the nucleotides between −1009 and −504 bp were deleted. Further deletions from −504 to −197 resulted in decreased promoter strength. The highest GUS activity was observed in the construct pC, which contained the promoter fragment from −504 to −1 bp (Fig. 1). In the transgenic plants, the 5’-deletion Gacab promoter fragments A, B, and D (Fig. 1) had relative activities of 50.2%, 60.0%, and 51.5%, respectively, compared to the construct pC. The −504 to −1 bp (C) fragment of the Gacab promoter had an activity of 160%compared with

Fig. 2 Histochemical GUS assay of transgenic tobacco leaves A: Transgenic tobacco with pA; B: Ttransgenic tobacco with pB; C: Transgenic tobacco with pC; D: Transgenic tobacco with pD; E: Transgenic tobacco with pBI121; F: Control NC89.

CaMV35S. The strength of the other Gacab promoter deletion constructs was similar to that of CaMV35S (Fig. 3). These results were in agreement with the transient GUS expression in rice calli [15]. 2.3 Analysis of light inducibility of Gacab promoter constructs in transgenic plants When seedlings transferred from the dark to the light condition, the expression of construct pA was not detected in the dark-growing seedlings, whereas its expression was measurable for seedlings grown in the light. Gene gus was expressed throughout the seedlings containing pB, pC, or pD grown either in the dark or in the light (Fig. 4).

GUS activity (pmol min−1 mg−1)

WANG Xu-Jing et al. / Acta Agronomica Sinica, 2009, 35(6): 1006–1012

Fig. 3

Gacab promoter deletion analysis in transgenic plants expressing gus reporter gene Data are means of 3 independent experiments for each construct, and 20 independent lines for each construct were assayed.

Fig. 4 Light inducible and tissue specific expression characteristic of Gacab promoter 5’ deletion mutant A: Transgenic tobacco containing Gacab promoter 5’ deletion mutant was geminated and grown in the dark. Gus expression was detected in the whole plant of transgenic tobacco containing pB, pC, pD, and pBI121, but not in the control and the transgenic plant containing pA. B: Plants were grown in the light for 2 d after germinated in the dark. All transgenic plants expressed GUS effectively. Transgenic plants containing pA only expressed GUS in green tissue, and others expressed GUS in all tissues. C: Transgenic tobacco were germinated and grown in the light. All transgenic plants expressed GUS effectively. Transgenic plants containing pA only expressed GUS in green tissue, and others expressed GUS in all tissues. In all panels, a: Transgenic tobacco containing pA; b: Transgenic tobacco containing pB; c: Transgenic tobacco containing pC; d: Transgenic tobacco containing pD; e: Transgenic tobacco containing pBI121; f: NC89, the control.

WANG Xu-Jing et al. / Acta Agronomica Sinica, 2009, 35(6): 1006–1012

3

Discussion

Generally, positive and negative regulatory elements are contained in the light-inducible promoters that control gene transcription, and it is assumed that gene expression is controlled by combinatorial regulation of transcriptional elements. Upstream sequences that promote gene expression have been observed in cab from pea [18] and N. plumbaginifolia [12]. Ha and An [10] found that the −321 to −253 bp region upstream of Arabidopsis cab1 enhances cab1 promoter activity. Cecillia et al. [7] found that a 268 bp DNA fragment (−357 to −89 bp) of the wheat cab promoter is an enhancer and comprises of 3 elements. According to expression strengths of the Gacab promoter deletion constructs in transgenic plants in this study, the positive regulatory elements,

which could enhance gene expression, exist from −504 to −197 bp; whereas, the negative elements are located from −1009 to −504 bp. On the basis of PlantCARE analysis, the following elements are found in the Gacab promoter: 2 GT1 elements in the region from −802 to −504 bp (5’-GGTTAA-3’ and 3’-TAGTTGG-5’), 1 5’-UTR pyrimidine-rich stretch in the −498 bp region (5’-TTTCTCTCTCTCTC-3’), 1 Sp1 (5’-TTT CTCTCTCTCTCCC-3’), and 6 CAAT boxes from −504 to −262 bp (Fig. 5). The GT1 motif, which is responsible for inhibiting transcription of light-inducible genes in dark conditions [19], is located at −779 to −504 bp and may be used as a negative element to modulate the expression strength of the promoter. The 5’-UTR pyrimidine-rich region is a cis-acting element that confers high transcription levels [20]. Spl plays an

Fig. 5 Sequence analysis of Gacab promoter Upper strand is “+”strand, and the lower one is “í” strand. “A” of initiation codon ATG is the “+1” position. ATG and TATA box are underlined. G-box and I-box are shaded in black and gray, respectively. GT1 element is framed and shaded in gray. CAAT box is framed. 5’-UTR pyrimidine-rich stretch is shaded in gray. SP1 is underlined and shaded.

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important role in transcription activation [21]. The CAAT boxes are common cis-acting elements in promoter and enhancer regions [22]. Given knowledge on these promoter elements, the −504 to −197 bp region likely enhances the strength of Gacab promoter. This hypothesis is supported by GUS activity results, where the highest expression was observed using the pC construct. In this study, only the full-length Gacab promoter proves to be light inducible, and several light-responsive elements are present between í1009 and í779 bp. In fact, this region harbors several potential light-responsive and organ-specific regulatory elements according to sequence analysis, including sequences similar to the ubiquitous light-regulated elements (LREs) in light inducible promoters, I-box and G-box. These elements are indispensable in light regulated transcription activation [23–25].

4

[4]

[5]

[6]

[7]

[8]

Conclusions

In this study, plant expression vectors was constructed, in which gus (uid A) gene was driven by the full-length Gacab promoter and 5’ truncations with the lengths of 197, 504, and 779 bp. All constructs were transformed into tobacco. With the help of gus expression system in the transgenic tobacco plants, only the full-length Gacab promoter proves to be lightinducible and specifically expressed in green organs. The promoter fragment from í504 to í1 bp has the highest activity of GUS expression, which is 0.6-fold higher than that of the CaMV35S promoter.

[9]

[10]

[11]

[12]

Acknowledgments This study was supported by the National High Technology Research and Development Program of China (2007AA100505 and 2007AA10Z182), the National Natural Science Foundation of China (30671337), and the key project for Science and Technology Development on New Varieties of Genetic Modified Organisms (2008ZX08005-003).

[13]

[14]

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