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Keywords:

  • cell-free protein synthesis;
  • cellulosome;
  • Clostridium thermocellum

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References
  9. Supporting Information

Endoglucanase CelJ (Cel9D-Cel44A) is the largest multi-enzyme subunit of the Clostridium thermocellum cellulosome and is composed of glycoside hydrolase (GH) families 9 and 44 (GH9 and GH44) and carbohydrate-binding module (CBM) families 30 and 44 (CBM30 and CBM44). The study of CelJ has been hampered by the inability to isolate full-length CelJ from recombinant Escherichia coli cells. Here, full-length CelJ and its N- and C-terminal segments, CBM30-GH9 (Cel9D) and GH44-CBM44 (Cel44A), were synthesized using a wheat germ cell-free protein synthesis system and then were purified to homogeneity. Analysis of the substrate specificities of CelJ and its derivatives demonstrated that the fusion of Cel9D and Cel44A results in threefold synergy for the degradation of xyloglucan, one of the major structural polysaccharides of plant cell walls. Because CelJ displayed broad substrate specificity including significant carboxymethylcellulase (CMCase) and xylanase activities in addition to high xyloglucanase activity, CelJ may play an important role in the degradation of plant cell walls, which are composed of highly heterogeneous polysaccharides. Furthermore, because Cel9D, but not Cel44A, acts as a semi-processive endoglucanase, the different modes of action between Cel9D and Cel44A may be responsible for the observed synergistic effect on the activity of CelJ (Cel9D-Cel44A).


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References
  9. Supporting Information

Clostridium thermocellum is an anaerobic thermophilic cellulolytic bacterium that rapidly degrades crystalline cellulose and secretes a wide variety of polysaccharide-degrading enzymes and structural proteins. In the presence of calcium ions, these secreted protein factors, which include cellulases, hemicellulases, and scaffolding proteins, are spontaneously assembled into a supramolecular multi-enzyme complex termed a ‘cellulosome’ that is displayed on the C. thermocellum cell surface (Felix & Ljungdahl, 1993). Cellulosome formation is mediated by species-specific interactions between the dockerin and cohesin modules of enzymatic and scaffolding subunits, respectively. The C. thermocellum cellulosome has a mass of 2000–6500 kDa, although a larger complex, called a ‘polycellulosome’, with a mass of 50 000–80 000 kDa is also formed. The efficient degradation of recalcitrant crystalline cellulose by C. thermocellum is essentially dependent on the formation of a cellulosome complex containing the primary scaffolding protein CipA, which displays nine cellulosomal components through cohesin–dockerin interactions. Analyzing the cellulolytic mechanism of the cellulosome is beneficial to the development of enzymatic processes that efficiently breakdown cellulosic biomass into soluble sugars, which are used as fermentation substrates for the production of renewable bio-based fuels and chemicals (Jarboe et al., 2010).

The genomic DNA of C. thermocellum ATCC27405 contains at least 86 cellulosomal genes, 12 of which are predicted to encode scaffolding proteins due to the presence of cohesin module(s) and the remaining 74 are likely to encode cellulosomal components containing a dockerin module for binding to scaffolding proteins. Among these predicted dockerin-containing cellulosomal proteins, the 178-kDa endoglucanase CelJ (Cel9D-Cel44A), is the major and largest enzymatic subunit in the C. thermocellum cellulosome (Ahsan et al., 1996; Gold & Martin, 2007; Raman et al., 2009). CelJ consists of 1601 amino acids (a.a.) and contains glycoside hydrolase (GH) families 9 and 44 (GH9 and GH44) and carbohydrate-binding module (CBM) families 30 and 44 (CBM30 and CBM44) (Fig. 1a). The enzymatic characteristics of individual modules of CelJ have been investigated in detail to determine the role of CelJ in the hydrolysis of plant cell-wall polysaccharides. Specifically, the substrate specificities and binding properties of the GH9, GH44, CBM30, CBM44, CBM30-GH9 (designated as Cel9D), and GH44-CBM44 (designated as Cel44A) modules have been determined (Ahsan et al., 1997; Arai et al., 2003; Najmudin et al., 2006) and, in addition, the crystal structures of the CBM30, CBM44, and GH44 modules were solved (Najmudin et al., 2006; Kitago et al., 2007). Despite these findings, the activity of full-length CelJ remains unclear because it is difficult to isolate full-length protein from recombinant Escherichia coli cells due to endogenous proteolysis (Ahsan et al., 1996). Thus, determining the mechanism of CelJ activity requires the development of a method to express and purify full-length CelJ. Furthermore, the combination of cellulases with different enzymatic activities often generates substantial synergistic activity for the degradation of polysaccharides. CelJ is formed by the fusion of Cel9D and Cel44A, which are both classified as endoglucanases and exhibit broad substrate specificity but have different modes of action, with Cel9D acting as a semi-processive endoglucanase (Ahsan et al., 1997; Arai et al., 2003; Najmudin et al., 2006). However, it is not clear whether the combination of Cel9D and Cel44A provides synergy for polysaccharide degradation by CelJ. Thus, analyzing the activity of full-length CelJ reveals the synergy for polysaccharide degradation imparted by the fusion of Cel9D and Cel44A.

image

Figure 1. Domain organization and SDS-PAGE analysis of purified CelJ and its deletion derivatives, Cel9D and Cel44A. (a) Clostridium thermocellum cellulosomal cellulase CelJ, the largest multi-enzyme subunit constituting the cellulosome, contains two glycoside hydrolases, GH9 and GH44, and two carbohydrate-binding modules, CBM30 and CBM44. The N-terminal segment of CelJ, designated as Cel9D, is composed of CBM30, an Ig-like domain, and GH9, and the C-terminal segment, designated as Cel44A, is composed of GH44, a type-I dockerin domain, a polycystic kidney disease (PKD) domain, and CBM44. Mature CelJ and its deletion derivatives, Cel9D and Cel44A, were cell-free synthesized as GST-fusion proteins containing a FLAG tag at the C-terminus, and then purified by cleavage with PreScission protease in a column. (b) Purified samples were subjected to SDS-PAGE on a 10% gel and stained with Coomassie brilliant blue. The bands corresponding to the purified CelJ, Cel9D, and Cel44A proteins are indicated by asterisks. (c) Full-length CelJ and its deletion derivatives were detected by Western blotting analysis with an anti-FLAG M2 monoclonal antibody that targeted the C-terminal FLAG tag of the proteins.

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Here, we synthesized and purified full-length CelJ using a wheat germ cell-free protein synthesis system that is suitable for producing large multi-domain proteins (Hirano et al., 2006a). Analysis of the substrate specificities of purified CelJ and its deletion derivatives revealed the synergy for the degradation of cellulosic substrates generated by the fusion of Cel9D and Cel44A.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References
  9. Supporting Information

Materials

The genomic DNA of C. thermocellum NBRC103400 (ATCC27405) was obtained from the National Institute of Technology and Evaluation, Japan, and its nucleotide sequence is publicly available in the National Center for Biotechnology Information database under accession no. NC_009012.1. Plasmid pEUGFP, which was used for the cell-free protein synthesis of green fluorescent protein (GFP), was constructed previously (Hirano et al., 2006a), plasmid pGEX-6P-1 was purchased from GE Healthcare, and wheat germ extract was purchased from Cell-Free Science, Japan. Escherichia coli DH5α (Takara, Japan) was used as a cloning host. All PCR primers were synthesized by Greiner Japan and are listed in Table S1 (Supporting Information).

DNA substrate for cell-free protein synthesis of GST-CelJ fusion protein

Plasmid pEUGST-GFP, a cassette vector for the construction of pEU derivatives for the cell-free protein synthesis of a glutathione S-transferase (GST) fusion protein, was constructed as follows. A DNA fragment encoding a GST-GFP fusion protein was constructed by first PCR-amplifying the GST gene in pGEX-6P-1 with primers GST-5′ and GST-CPr3′ and the GFP gene in pEUGFP with primers GFP-NPr5′ and GFP-3′. The two amplified DNA fragments containing the GST and GFP genes, respectively, were purified by gel electrophoresis, combined, and subjected to a second round of PCR using primers GST-5′ and GFP-3′. After digesting the fused PCR product with NheI and SalI, the DNA fragment was ligated into the SpeI-SalI sites of pEUGFP to yield pEUGST-GFP, which was then transformed into DH5α cells. Positive transformants were selected on Luria–Bertani (LB) agar plates containing 50 μg mL−1 ampicillin (Amp; LB-Amp plates), and incubated at 37 °C.

Plasmid pEUGST-CelJ for the cell-free protein synthesis of GST-CelJ fusion protein was constructed as follows. The CelJ gene was PCR-amplified from C. thermocellum NBRC103400 genomic DNA using primers CelJ-NPr5′ and CelJ-CF3′. After digestion with BglII and SalI, the amplified DNA fragment was introduced into the BamHI-SalI sites of pEUGST-GFP to exchange the GFP and CelJ genes, yielding plasmid pEUGST-CelJ, which was then transformed into DH5α cells. After the selection of positive transformants on LB-Amp plates incubated at 37 °C, the nucleotide sequence of the CelJ gene in pEUGST-CelJ was confirmed by the dideoxy chain termination method with fluorescent dye terminators using an ABI PRISM 310 sequencer (Perkin-Elmer, Japan). The DNA substrate for cell-free protein synthesis of the GST-CelJ fusion protein was PCR amplified from pEUGST-CelJ using primers pEUUn and 2pEUDn.

PCR was performed for 25 cycles using the following conditions: 96 °C for 30 s, 50 °C or 55 °C for 30 s, and 72 °C for 60 s per 1 kbp with PrimeSTAR GXL or HS DNA polymerase (Takara) using a Takara PCR Thermal Cycler Dice TP-650.

DNA substrates for cell-free protein synthesis of GST-Cel9D and GST-Cel44A fusion proteins

The DNA substrate for the cell-free protein synthesis of the GST-Cel9D fusion protein was constructed as follows. A DNA fragment consisting of the 5′ UTR-GST-Cel9D region in pEUGST-CelJ, which also contained the promoter sequence of SP6 RNA polymerase and the omega sequence, was amplified by PCR with primers pEUU and Cel9D-CF3′. The pEUGST-CelJ region containing the 3′ UTR of tobacco mosaic virus was also amplified by PCR using primers pEU-CF5′ and pEUD. The two amplified DNA fragments were purified by gel electrophoresis, combined (each at a final concentration of 5 nM), and subjected to a second round of PCR without primers.

The DNA substrate for the cell-free protein synthesis of the GST-Cel44A fusion protein was constructed as follows. The region encompassing 5′ UTR-GST in pEUGST-CelJ was amplified by PCR with primers pEUU and GST-Pr3′, and the region encompassing Cel44A-3′ UTR in pEUGST-CelJ was amplified by PCR with primers Cel44A-Pr5′ and pEUD. The two amplified DNA fragments were purified by gel electrophoresis, combined (each at a final concentration of 5 nM), and subjected to a second round of PCR without primers.

The DNA fragments generated by the second round of PCR were subjected to a third round of PCR with primers pEUU and pEUDn. As the DNA yield of the third-round PCR was low, the amplicons were further amplified by a fourth round of PCR with primers pEUUn and 3pEUDn and the resulting products were used as a DNA substrate for the cell-free protein synthesis of the GST-Cel9D and GST-Cel44A fusion proteins.

Cell-free protein synthesis and purification

The wheat germ cell-free protein synthesis and purification of the GST fusion proteins were performed as described previously (Hirano et al., 2006b). The synthesized fusion proteins were cleaved by PreScission protease in a glutathione-Sepharose 4B MicroSpin column (GE Healthcare). The flow-through fraction contained proteins of the predicted sizes for FLAG tag-fused mature CelJ (176 kDa), Cel9D (84 kDa), and Cel44A (93 kDa) proteins, as revealed by SDS-PAGE on 10% gels stained with Coomassie brilliant blue and Western blot analysis using an anti-FLAG M2 monoclonal antibody (Sigma-Aldrich, Japan) with an ECL Advanced Detection kit (GE Healthcare). The protein concentration of the CelJ, Cel9D, and Cel44A proteins was estimated by densitometric analysis with image j software (National Institute of Health) and the use of bovine serum albumin (BSA) as a standard.

Cellulase assay

Cellulolytic activity was assayed by the incubation of purified enzymes and cellulosic substrates for 30 min at 55 °C in a 10-μL reaction mixture containing 50 mM sodium acetate (pH 5.5), 2 mM CaCl2, 2 mM dithiothreitol, and 0.01% BSA. As assay substrates, microcrystalline cellulose (Avicel PH-101; Sigma-Aldrich), phosphoric acid-swollen cellulose (PASC) prepared from Avicel, as described previously (Wood, 1988), CMC (Sigma-Aldrich), xylan from beech wood (Wako, Japan), and xyloglucan from tamarind (Megazyme, Ireland) were used at a final concentration of 5 mg mL−1 (0.5%). For cellulase assays against recalcitrant substrates, the incubation time was extended from 30 min to 2 or 18 h. The amount of reducing sugars released from the substrate was determined with 3′,5′-dinitrosalicylic acid (DNS) reagent, as described previously (Miller, 1959). Briefly, after completion of the incubation period, DNS reagent (10 μL) was added to each reaction mixture, which was then heated at 95 °C for 5 min. After 2.5- or 5-fold dilution with water, the concentration of reducing sugars was quantified by measurement of absorbance at 535 nm with the use of glucose as a standard. One unit of enzymatic activity was defined as the amount of enzyme producing 1 μmol of reducing sugar per minute, and specific activity was defined as the enzymatic activity per μmol of enzyme. Assays were performed with various amounts of enzyme to examine if the amount of product increased in proportion to that of the enzyme. Data are presented as the means from at least three independent experiments, and the SD are within 40% for values reported.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References
  9. Supporting Information

Cell-free synthesis and purification of CelJ

To analyze the activity of full-length CelJ for various polysaccharides, we constructed plasmids encoding full-length CelJ and the N- and C-terminal segments of CelJ, Cel9D and Cel44A, respectively, which contain the GH9 and CBM30 modules and the GH44 and CBM44 modules, respectively (Fig. 1a). In previous studies, the CelJ deletion derivatives Cel9D and Cel44A were purified to homogeneity (Ahsan et al., 1997; Arai et al., 2003; Najmudin et al., 2006), although the purification of full-length CelJ from recombinant E. coli cells was unfeasible as a result of endogenous proteolysis (Ahsan et al., 1996). However, here, we attempted to synthesize full-length CelJ and its deletion derivatives as GST-fusion proteins by in vitro translation using a wheat germ cell-free protein synthesis system. The synthesized fusion proteins were recovered in soluble form, purified to homogeneity by glutathione affinity chromatography, and cleaved to yield the full-length and deletion derivatives (Fig. 1b). Purification of the full-length form of CelJ, which was 176 kDa in size, was confirmed by SDS-PAGE (Fig. 1b) and Western blotting analysis using an antibody against the C-terminal FLAG tag (Fig. 1c). As estimated by SDS-PAGE and densitometric analyses, the amount of CelJ purified from 1 mL of translation mixture was 10 μg.

Substrate specificity of CelJ

The enzymatic properties of Cel9D and Cel44A, such as optimal pH and temperature, and substrate specificities have been reported in detail (Ahsan et al., 1997; Arai et al., 2003; Najmudin et al., 2006). Cel9D has a high activity for CMC and a low activity for acid-swollen cellulose (ASC) and Avicel, but displays no detectable activity towards oat spelt xylan (Arai et al., 2003). In contrast, although Cel44A also exhibits high and low activities for CMC, and ASC and Avicel, respectively, it has significant activity for oat spelt xylan (Ahsan et al., 1997; Najmudin et al., 2006). These results are reasonably consistent with their observed substrate binding properties; the carbohydrate-binding module of Cel9D (CBM30) binds to CMC, ASC, and Avicel, but not to oat spelt or birch wood xylan (Arai et al., 2003; Najmudin et al., 2006), whereas that of Cel44A (CBM44) binds to CMC, ASC, Avicel, and oat spelt xylan (Najmudin et al., 2006). Moreover, Cel44A displays a high activity for xyloglucan (a highly branched β-1,4-glucan), which was shown to bind CBM44 (Najmudin et al., 2006). Although the xyloglucanase activity of Cel9D has not been reported, CBM30 binds to xyloglucan with lower affinity than that of CBM44 (Najmudin et al., 2006). The substrate specificity of full-length CelJ was previously investigated using a crude extract obtained from CelJ-expressing recombinant E. coli cells, and both CMCase and xylanase activities were detected by zymogram analysis of the cell lysate (Ahsan et al., 1996). Prior to the present study, however, the activity of purified CelJ had not been quantified.

Here, we examined the substrate specificities of full-length CelJ and its deletion derivatives by measuring the hydrolytic activities of these purified proteins for various cellulosic substrates, including Avicel, PASC, CMC, xylan, and xyloglucan. In addition, we analyzed the synergy of CelJ for the degradation of these cellulosic substrates by comparing the specific activities per μmol between CelJ and an equimolar mixture of Cel9D and Cel44A (Cel9D+Cel44A) (Table 1). Cel9D showed a high activity for CMC and xyloglucan, but no detectable activity for Avicel, PASC, or xylan, whereas Cel44A exhibited relatively high activity for CMC, xylan, and xyloglucan, but had no detectable activity towards Avicel or PASC. Although our assay system showed a high background level (2.5 U μmol−1), these results were comparable to the previous findings for Cel9D and Cel44A, as described above (Ahsan et al., 1997; Arai et al., 2003; Najmudin et al., 2006). In contrast, CelJ showed a high activity for CMC, xylan, and xyloglucan, a low activity for PASC, and no detectable activity for Avicel. Notably, the equimolar mixture of Cel9D and Cel44A had no detectable activity for PASC (< 2.5 U μmol−1), in contrast to full-length CelJ, which showed at least fourfold higher activity for this substrate (10 U μmol−1), indicating that the fusion of Cel9D and Cel44A results in synergy for PASC degradation. Similarly, the fusion of Cel9D and Cel44A also generated synergy for xylan degradation.

Table 1. Enzymatic activities of CelJ and its deletion derivatives for various cellulosic substrates
SubstrateSpecific activity (U μmol−1)
Cel9DCel44ACel9D+Cel44ACelJ
  1. The enzymatic activities of Cel9D, Cel44A, and CelJ were determined by measurement of the amount of reducing sugars released from the substrate (0.5%) as described in Materials and methods. An equimolar mixture of Cel9D and Cel44A is designated as Cel9D+Cel44A. The theoretical activities of Cel9D+Cel44A, which were determined by summing the individual activities of Cel9D and Cel44A, are indicated in parentheses after the apparent activities for CMC, xylan, and xyloglucan. Data are presented as the means of at least three independent experiments, and SD are within 40% for values reported.

  2. a

    Activity did not exceed the background level (< 2.5 U μmol−1).

Aviceaaaa
PASCaaa10
CMC8878480(166)770
Xylana6258(62)113
Xyloglucan4922781018(770)3070

Among the examined substrates, CelJ showed the highest specific activity for xyloglucan degradation, a finding that is attributable to the substantial synergy generated by the fusion of Cel9D and Cel44A. Specifically, the apparent enzymatic activities of Cel9D+Cel44A for CMC and xyloglucan were 2.9- and 1.3-fold higher, respectively, than the theoretical activities, which were determined by summing the individual activities of Cel9D and Cel44A (480 vs. 166 U μmol−1 for CMC, and 1018 vs. 770 U μmol−1 for xyloglucan). The activities of CelJ were 1.6- and 3.0-fold higher for CMC and xyloglucan, respectively, than those of Cel9D+Cel44A (770 vs. 480 U μmol−1 for CMC, and 3070 vs. 1018 U μmol−1 for xyloglucan). These results indicate that the combination of Cel9D and Cel44A generated more synergy for CMC degradation than for xyloglucan degradation, whereas the physical fusion of Cel9D and Cel44A generated more synergy for xyloglucan degradation than for CMC degradation.

Based on our analyses, CelJ has 4.0-fold higher specific activity for xyloglucan (3070 U μmol−1) than for CMC (770 U μmol−1) (Table 1). However, CelJ cannot be classified as a specific xyloglucanase, such as C. thermocellum Xgh74A (Zverlov et al., 2005; Martinez-Fleites et al., 2006), because specific xyloglucanases are defined as enzymes that exhibit at least 10-fold higher activity for xyloglucan than for CMC (Grishutin et al., 2004), and CelJ did not meet this criterion. Furthermore, although CelJ was previously identified by proteomic analysis as a major enzymatic subunit of the C. thermocellum ATCC27405 cellulosome obtained from cells grown on crystalline cellulose (Gold & Martin, 2007; Raman et al., 2009), the proportion of CelJ increased when the carbon source was changed from crystalline cellulose (i.e. homogeneous cellulose) to pretreated switchgrass (i.e. highly heterogeneous polysaccharides), as is commonly observed for hemicellulases (e.g. xylanases or xyloglucanases) (Raman et al., 2009). Thus, the broad substrate specificity exhibited by CelJ may play an important role in the degradation of plant cell walls composed of highly heterogeneous polysaccharides.

Although it remains unclear why synergy for the degradation of CMC and xyloglucan in particular is generated upon the fusion of Cel9D and Cel44A, the different modes of Cel9D and Cel44A activities upon hydrolysis may be an underlying factor. CelJ is predicted to interact with CMC and xyloglucan through CBM44-mediated binding, because C-terminal CBM44 shows 10-fold higher binding affinity to these substrates than N-terminal CBM30 (Najmudin et al., 2006). The GH44 module adjacent to CBM44 cleaves CMC and xyloglucan at the internal β-1,4-glucosidic bond (Ahsan et al., 1997; Najmudin et al., 2006). Following cleavage of this bond, Cel44A introduces the neighboring Cel9D to the reducing end of this cleavage site, and the introduced Cel9D proceeds to hydrolyze the chain from the reducing end. Cel9D is proposed to function as a semi-processive endoglucanase because the enzyme reduces the viscosity of a CMC solution at a slower rate than C. thermocellum CelC, a typical endoglucanase (Arai et al., 2003). This finding suggests that Cel9D cleaves CMC at the internal β-1,4-glucosidic bond and proceeds to hydrolyze the substrate from the reducing end of the cleavage site with low processivity. In fact, the combination of Cel9D and CelC generated clear synergy for ASC degradation (Arai et al., 2003), supporting the notion that Cel9D and Cel44A act synergistically. Moreover, a similar synergistic effect on cellulolytic activity generated by the fusion of endo- and exo-glucanases has been reported (Riedel & Bronnenmeier, 1998); the artificial fusion of GH9 processive endoglucanase CelZ and GH48 exoglucanase CelY from Clostridium stercorarium showed twofold synergy for the degradation of Avicel as compared with the equimolar mix of these cellulases, termed ‘intramolecular synergism’. Further studies on the mode of CelJ action are needed to reveal the mechanism underlying the synergistic action between Cel9D and Cel44A in full-length CelJ.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References
  9. Supporting Information

We thank S. Nakayama for technical support. This work was supported by JST, PRESTO, a Grant-in-Aid for Young Scientists (B) 22760612 from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Research Grant from the College of Engineering, Nihon University.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Authors' contribution
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
fml12149-sup-0001-TableS1.docWord document34KTable S1. Primers used for DNA amplification.

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