Mol Biol Rep DOI 10.1007/s11033-014-3309-0

Cloning and analysis of a functional promoter of fungal immunomodulatory protein from Flammulina velutipes Wei-Ran Cong • Yan Liu • Qi-Zhang Li Xuan-Wei Zhou

Received: 5 September 2013 / Accepted: 14 February 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Fugal immunomodulatory protein from Flammulina velutipes (FIP-fve) belongs to FIPs family, which has precious pharmaceutical value. To understand the regulatory mechanism of FIP-fve expression, we have cloned a 900 bp genomic DNA fragment from the transcriptional start site of the FIP-fve gene using genomic walker technology. Sequence analysis showed the presence of several eukaryotic transcription factor binding motifs in the 900 bp of upstream region of the FIP-fve gene, which contains one putative TATA-boxes, four possible CAAT-boxes, one ABRE, one ARE, three CGTCA-motifs, two TGA-elements and four Skn1 motifs. The eukaryotic expression vector pfveP:: GUS-GFP was transferred into tobacco via an agrobacterium-mediated leaf disc transformation. The results showed that the FIP-fve promoter could induce the reporter gene GUS or GFP expression in different tissues of tobaccos. This study would lay a foundation for expression regulation of FIP-fve and development of genetic-modified plant products. Keywords Fungal immunomodulatory protein (FIP)  Flammulina velutipes  Promoter  Transgenic tobacco  Function Abbreviations FIP Fungal immunomodulatory protein ACCC Agricultural Culture Collection of China PDA Potato dextrose agar BDGP Berkeley Drosophila Genome Project

W.-R. Cong  Y. Liu  Q.-Z. Li  X.-W. Zhou (&) Shanghai Key Laboratory of Agri-biotechnology, Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, No. 800, Dongchuan Road, 200240 Shanghai, People’s Republic of China e-mail: [email protected]


Cis-acting regulatory element Luria-Bertani Abscisic acid Methyl jasmonate Fungal immunomodulatory protein from F. velutipes

Introduction Flammulina velutipes (Curt.:Fr.) Sing, named as Enoki mushroom in English and golden needle mushroom in Chinese, belongs to Flammulina, Tricholomataceae, Agaricales. The fungus is also known as winter mushroom or velvet stems with worldwide distribution. This mushroom is mainly cultivated for gourmet and tonic purposes. It is not only of high nutritional values, but also of various medicinal components, so it is popular in Eastern Asian countries, especially in China, Japan, and South Korea [1]. Up to now, various bioactive compounds have been isolated from F. velutipes, including polysaccharides, protein-glucan complex, sterols, lectins, peroxidases, laccases, cellulases, proteases and so on. These bioactive compounds present different medicinal and pharmaceutical properties [2, 3]. Among these bioactive components, polysaccharides have been paid a lot of attentions [2, 4–6], and fungal proteins have also attracted quantities of researchers in recent years [7–10]. Fungal immunomodulatory protein (FIP), isolated from the fruiting bodies of higher basidiomycetes, are a kind of small molecule protein with immune regulating activity. Kino et al. isolated the first FIP from the fruiting bodies of Ganoderma lucidum and named it as Ling Zhi-8 (LZ-8 or FIP-glu) in 1989 [11]. There are eleven FIPs have been isolated and identified up to now. All these proteins make up new protein family-FIPs, which include FIP-glu, FIP-gts,


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FIP-fve, FIP-gja, FIP-vvo, FIP-gsi, FIP-gmi, FIP-gas, FIPtvc, FIP-SN, FIP-SJ [12]. FIPs among this family are composed of 110–114 amino acids with high structural similarity and 60–70 % homology [13]. However, different members in FIPs family differ not only in amino acid sequence, but also in degrees of immune function in vivo and in vitro [13, 14]. FIP-fve was firstly isolated from F. velutipes and confirmed its complete amino acid sequence in 1995 [15]. The FIP-fve showed that it was able to hemagglutinate human red blood cells, stimulate blast-forming activity of human peripheral blood lymphocytes and gene expression of interleukin-2, interferon-gamma and tumor necrosis factor-alpha [16, 17]. However, the yields of FIP-fve from natural mushroom are low [11, 18, 19], many researchers have to improve the yield of FIPs by means of genetic engineering methods, so that the production of FIPs could come into industrial process. Scientists worldwide focused their interests on utilizating genetic engineering technology to develop FIP’s products, such as cloning the FIP genes, expression of the genes in prokaryotic and eukaryotic cells, and how to enhance the bioactivities of FIPs [20]. For this process, a suitable promoter is very important for successful expression of exogenous gene. Based on their functions, promoters in genetic engineering can be divided into three types: constitutive promoter, tissue-specific promoter and inducible promoter. Different types of promoters have different functions for various hosts or expression of products. The objective of this study is to clone the promoter of FIP-fve using genomic walking techniques, and to construct the plant binary expression vector and transfer tobacco in order to identify the function of this promoter. Further, we hope this research will lay a foundation for the regulation of FIP-fve gene expression and development of genetic-modified plant products.

Materials and methods Microorganisms and materials Flammulina velutipes was purchased from the Agricultural Culture Collection of China (ACCC) (Beijing, China). Nicotiana tabacum seeds, Esherichia coli strains DH5a, Agrobacterium tumefaciens strains EHA105 and pCAMBIA1304 plasmid were preserved by Plant Biotechnology Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, China.

were obtained from the margins of actively growing hypha and inoculated into a 250 mL Erlenmeyer flask containing the same medium without agar (liquid medium). Cultures were maintained on a rotary shaker at 120 rpm at 28 °C for about 5 d and harvested by centrifugation at 5,000 9 g for 15 min. After being washed twice with asepsis water, the mycelia were collected by centrifugation at 3,000 9 g for 15 min for extraction of genome DNA. Cloning of FIP-fve promoter The DNA extraction was using the high salt, low pH method (HSLPM) as described in previous publication [21]. The Genome Walker DNA libraries were constructed using the Universal Genome WalkerTM Kit (Clontech, USA). The genome DNA was digested by restriction enzymes EcoRI, HindIII, PstI, SalI and XbaI, and then the enzyme-digested products were purified and ligated to adaptors at both ends by T4 DNA ligase (TaKaRa, China). The 50 upstream promoter region of FIP-fve was used to conduct two PCR amplifications using these four DNA libraries as templates. The primary PCR used gene specific primers S1: 50 -TAGCTG CTTGGGGTACCACGGCC-30 designed according to the cDNA sequence of FIP-fve gene (Gene Bank: GU388420) and AP1: 50 -GTAATACGACTCACTATAGGGC-30 (kit provided adaptor primer), and the product was afterward diluted and used as the template in the secondary PCR using primer pairs of S2: 50 -AAGGTGAGCGACGTGGCGGACAT-30 and AP2: 50 -ACTATAGGGCACGCGTGGT-30 (kit provided adaptor primer). The gene fragments were amplified by PCR and the products were purified using Gel Extraction Mini Kit (Watson, China), ligated to pMD18-T vectors (TaKaRa, Dalian, China), transformed into E. coli. The transcription start site was analyzed using Neural Network Promoter Prediction ( promoter.html). The PLANTCARE database (http://bioin was used for promoter nucleotide sequence analysis. Construction of expression vector To analyze promoter activity, the 50 upstream promoter region of FIP-fve was constructed in the HindIII/NcoI site of the pCAMBIA1304 vector (CAMBIA, Canberra, Australia) by replacing CaMV35S promoter. Transformation of tobacco plant

Preparation of fungal mycelia The mycelia of F. velutipes stock were cultivated on potato dextrose agar (PDA, potatoes 200 g/L, dextrose 20 g/L, MgSO47H2O 1.5 g/L, KH2PO4 2.5 g/L, agar 15 g/L) medium at 28 °C, for 5–7 d. Two agar blocks (u 10 mm)


Eukaryotic expression vector pfveP::GUS-GFP constructed was transformed into tobacco by the leaf disk transformationregeneration method as described previously [22]. Transformants were selected on MS medium with 0.85 % agar containing 0.1 mg/L a-naphthalene acetic acid (NAA),

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1.0 mg/L 6-benzyl aminopurine (6-BA), 250 mg/L carbenicillin disodium and 30 mg/L hygromycin, and regenerated on hormone-free MS medium containing 250 mg/L carbenicillin disodium at 26 °C. Molecular analysis of transgenic tobacco plants After 2–3 weeks planting, transgenic plants were verified with three primer pairs amplifying total DNA from leaf samples of transgenic and nontransgenic plants according to the CTAB method [23]. The FIP-fve promoter sequence specific primers fve-PF: 50 -ACGACGAGCAGACAGGACGAGA-30 and fvePR: 50 -GAGGACAGATAGTCACTGGAGC-30 were used to amplify a 398 bp fragment, which demonstrated the presence of the FIP-fve promoter transgene. The primers Hyg-F: 50 -GT CGAGAAGTTTCTGATCG-30 and Hyg-R: 50 -GTTTCC ACTATCGGCGAGTACT-30 were used to amplify a 960 bp fragment, which demonstrated the presence of the hygromycin gene. The primers GUS-F: 50 -GCTCTACACCACGCCG AACACCTG-30 and GUS-R: 50 -TCTTCAGCGTAAGGGTAATGCGAGGTA-30 were used to amplify a 483 bp fragment, which demonstrated the presence of the GUS transgene. The expression content of the transferred genes in regenerated tobacco was confirmed by RT-PCR. Leaves of the 6-week-old seedlings of five T1 lines were collected for RT-PCR analysis. Total RNA was isolated from approximately 200 mg leaf and root tissues using the Plant RNA Mini Kit (Watson Biotechnologies, Inc, China) and treated with the DNAase I (Promega, Madison, WI, USA) to remove any DNA contamination following the manufacturer’s instructions. The total RNA (1 lg) was reversely transcribed with the PrimeScriptÒ RT Master Mix Perfect Real Time kit (TaKaRa, China). First strand cDNA was used as template for PCR amplifications GUS specific primers described above. The tobacco GAPDH gene (AJ133422) was used as the internal control. The primers were NtGAPDH-F: 50 -GGAAAGTCCTACCAGCATTG-30 and NtGAPDH-R: 50 -ATCTATTGTCTCCCACGAAG-30 . Histochemical GUS staining of the leaf and root GUS staining assay was carried out as described [24]. Samples of leaf and root from the positive tobacco plants in PCR amplification were immersed in GUS staining buffer incubated in 37 °C overnight and de-stained in 75 % ethanol. The X-gluc was diluted 20 times with substrate solution containing 10–100 mM EDTA, 1–5 mM K3[Fe (CN)6],1–5 mM K4[Fe(CN)6], 0.1 % tritron X-100. GFP localization The confocal laser-scanning microscope (Zeiss 710, Germany) was used to monitor the GFP localization.

Fig. 1 Electrophoresis photos of promoter sequence amplification of FIP-fve gene. Lane 1 shows DNA marker DL2000 (TaKaRa Biotechnology Co. Ltd., Dalian, China); lanes 2–10 shows the amplification of promoter sequence; lanes 1A, 2A, 3A, 4A, 5A shows the amplification of second PCR; lanes 1B, 2B, 3B, 4B, 5B shows the amplification of primary PCR using digested genomic DNA with five restriction enzymes (EcoRI, HindIII, PstI, SalI and XbaI) (the arrowhead shows the promoter amplification sequence)

Laser in the wavelength of 543 nm was used to detect the spontaneous fluorescence of the chloroplast in tobacco mesophyll cells and laser in the wavelength of 488 nm was used to activate the fluorescence of GFP.

Results and discussion Cloning of FIP-fve gene promoter The genomic DNA was completely digested with five restriction enzymes (EcoRI, HindIII, PstI, SalI and XbaI) separately and the DNA fragments were then ligated separately to the GenomeWalker adaptor. The adaptor-ligated genomic DNA fragments were referred to convenience as GenomeWalker libraries. After the libraries have been constructed, the protocol takes just for PCR amplification. Following PCR amplification, the DNA fragment of upstream sequence showed many bands in different template in 1.0 % agarose gel (Fig. 1). There is a specific band of about 1,000 bp in lane 3 using HindIII digested genomic DNA as template. Sequence analysis showed that 23 bp of the fragments was overlapped to 50 end of FIP-fve ORF sequence. Analysis of FIP-fve gene promoter sequence Analysis of transcription start sites using Neural Network Promoter Prediction ( moter.html) found a core promoter sequence: CTCCAAT ATCTATATAAGCTCTCCCCACTTTCGTAGTTCA A C CAGCAACC (SCORE = 0.99). The box letter A shows the site of transcription start at -100 bp positions


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Fig. 2 The promoter sequence of FIP-fve. The initial transcriptional site was noted with an arrow and box. The putative TATA-Box, CAAT-Box were marked by shading, ABRE elements, TGA-element, Box-W1, Akn-1 motif, LTR, CGTCA-motif and ARE-box were labeled using underlined

upstream from the start codon ATG (Fig. 2). The analysis and comparison of the FIP-fve gene promoter sequence with published sequences were performed with Blasting

Fig. 3 Transgene structures and verification of transformants. Transgene structures for the pfveP::GUSGFP fusion and verification of transformants from T0regenerated tobacco plants by PCR. M: DL2000 Marker; lane 1 plasmid pfveP::GUS-GFP; lane 2–9: transgenic lines 2–9, lane 10: blank control (negative control)


on the NCBI server (, and it showed no same or similar sequence, so it is a new sequence not yet reported. Usually, the structures of 50 gene protome region of eukaryotes comprised four parts: the site of start transcription, TATA box, CAAT box and GC box. TATA boxes were generally located at -32 ± 7 bp positions upstream from the start of transcription. The consensus sequence for the TATA box was [T(CG)TATA (TA)A1–3(CT)A] that was important for eukaryotic transcription [25]. A TATA sequence was found to be located at -25 bp positions upstream from the start of transcription in FIP-fve, which might be important for the transcription control as well. The consensus sequence for TATA box was ATAT (Fig. 2). Four CAAT boxes were identified at the -33, -49, -605 and -747 bp positions upstream from the start of transcription (Fig. 2). The CAAT box is sometimes important for the efficiency of eukaryotic transcription [26]. Usually, CAAT boxes were found at the -77 ± 10 bp positions upstream from the start of transcription, although longer intervals have also been found. In addition to cis-acting element, some other elements were also identified in the promoter region of the FIP-fve, which included inducible elements by physiological and environmental factors, e.g. abscisic acid responsiveness (ABRE: CGTACGTGCA), MeJA-responsiveness (CGTCAmotif: CGTCA), auxin-responsive element (TGA-element: AACGAC), the anaerobic induction motif (ARE: TGGTTT), fungal elicitor responsive element (Box-W1: TTGACC); low-temperature responsiveness (LTR: CCGAAA), and the tissue or developmental stage specific factors, e.g. endosperm expression (Skn-1-like motif: GTCAT; GCN4 motif: CAAGCCA).

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Molecular analysis of transgenic tobacco plants We generated eight independent GUS and GFP-expressing lines. The genome DNA of transformed tobacco was extracted and detected whether it was the transgenic tobacco by using the method of PCR amplification. The GUS and GFP were placed under the control of the FIP-fve promoter, known to confer GUS-GFP expression of transgenes in plants (Fig. 3a). They were shown to harbor and express the GUS and GFP gene as demonstrated by genomic DNA amplification using FIP-fve promoter specific fve-PF and fve-PR, hygromycin primers F and R, and GUS primers F and R, respectively. The result of PCR

shown that 398, 960 and 483 bp fragments was amplified using fve-PF and fve-PR, hygromycin primers F and R, and GUS primers F and R, respectively (Fig. 3b). The expression content of the transferred genes in regenerated tobacco was confirmed using RT-PCR method (Fig. 4). GUS staining analysis The histochemical GUS assays to evaluate the activity of the promoter were conducted in three different lines of pfveP::GUS-GFP tobacco transformants. Results obtained with these lines were very similar. In agreement with the results of RT-PCR, it showed that leaf epidermal hair and roots turned blue, indicating a slight GUS activity (Fig. 5). Fluorescence detection

Fig. 4 Measurement of levels of GUS mRNA by RT-PCR. NtGAPDH was used as the internal control. Lanes 3, 4, 6, 8 and 9 shown transgene line 3, 4, 6, 8 and 9

Fig. 5 Histochemical localization of GUS activity in transgenic tobacco plants containing pfveP::GUS-GFP fusion. a Leaf epidermal hair of non-transgenic tobacco plant; b leaf epidermal hair of

Leaf epidermal hair was observed under UV light using a fluorescence microscope, and green fluorescence on the leaf epidermal hair could be seen (Fig. 6). The results of fluorescence detection indicated that the 1,000 bp sequence (FIP-fve-P) at upstream of FIP-fve initiation codon ATG,

transgenic tobacco plants; c root of non-transgenic tobacco plants; d root of transgenic tobacco plants


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Fig. 6 Fluorescence detection of leaf epidermal hair. a Leaf epidermal hair of non-transgenic tobacco plant; b leaf epidermal hair of transgenic tobacco plant

was enough to activate reporter gene GUS-GFP expressing in different issues of tobaccos.

Discussion Promoter is not only an important cis element in the regulation of gene expression, but also an important constituent part in the genetic engineering expression vector, and promoter cloning is also significant for constructing genetic engineering vectors and expressing proteins [27]. In the field of edible mushrooms, studies of promoter mainly concentrate on the utilization of strong promoters and homologous promoters. Regarding the low conversion rate of edible mushrooms, use of strong promoters contributes to improving conversion rate, and use of homologous promoters contributes to the identification of regulatory factors, reducing the methylation, and thus improving conversion rate [28]. At present, promoters isolated from edible mushrooms mainly include ras (rat sarcoma) gene [29, 30], gpd (glyceraldehydes-3-phosphate dehydrogenase) gene [31, 32], pri (primase) gene [33], gla (glucoamylase) gene [34], cel1 (cellulose-growth-specific) gene [35], cel2 and cel4 gene [36], spr1(serine proteinase) gene [37], trp1 (tryptophan synthetase) gene [38, 39], etc. In our study, FIP-fve gene promoter was cloned for the first time, and the sequence motif contained in FIP-fve gene promoter sequences was classified, and divided as following: plant hormone response element, such as ABRE, CGTCA motif and TGA-element, inducible transcribed by plant hormone (ABA, MeJA and auxin); stress control element, such as Box-W1, LTR inducible transcribed by fungal elicitor and low-temperature; tissue specific expression element, regulating and controlling the specifically expressed motif Skn1. In previous studies, we have cloned 501 bp at 50 upstream of FIP-gsi gene DNA sequence containing a TATA-box, a CAAT-box and a G-box [40].


Promoter function analysis methods include bioinformatical analysis and experimental analysis. The former predicts promoter sequence based on database-dependent prediction, while the latter determines promoter cis element and function, specific strategies including dot mutation, gel retardation assay, transient expression, transformation, and yeast one hybridization, etc. [41]. In this study, FIP-fve promoter validation was carried out with transforming tobacco, and it showed that the FIP-fve promoter induced leaf epidermal hair and root to express peculiarly. However, it needs further experimental verification to confirm whether it is related to the resistance and inducing endosperm to express peculiarly. An important issue in molecular biology is how to utilize mass accumulated part of gene sequences to clone full-length gene and its regulatory sequence, while promoter cloning offers a simple and efficiency method. Besides, more and more transgenic animals and plants appear as genetic engineering develops, and transgenic plant as bioreactor has been used to produce recombinant proteins for medicinal purposes. As the demand for biopharmaceuticals is expected to increase, transgenic plants have the potential to provide virtually unlimited quantities of proteins for use as tools in both human health care and the bioscience. This study provides a foundation for research on production of FIPs using transgenic plant as bioreactor and their commercial applications.

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Cloning and analysis of a functional promoter of fungal immunomodulatory protein from Flammulina velutipes.

Fugal immunomodulatory protein from Flammulina velutipes (FIP-fve) belongs to FIPs family, which has precious pharmaceutical value. To understand the ...
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