GENE-39661; No. of pages: 5; 4C: Gene xxx (2014) xxx–xxx
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The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana Gaoyang Zhang, Yujia Zhang, Jiantang Xu, Xiaoping Niu, Jianmin Qi ⁎, Aifen Tao, Liwu Zhang, Pingping Fang, LiHui Lin, Jianguang Su Key Laboratory for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China
a r t i c l e
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Article history: Received 25 September 2013 Received in revised form 27 February 2014 Accepted 3 May 2014 Available online xxxx Keywords: CcCCoAOMT RACE Jute Southern hybridization Arabidopsis
a b s t r a c t The Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is a key enzyme in lignin biosynthesis in plants. In this study we cloned the full-length cDNA of the Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) gene from jute using homology clone (primers were designed according to the sequence of CCoAOMT gene of other plants), and a modified RACE technique, subsequently named “CcCCoAOMT1”. Bioinformatic analyses showed that the gene is a member of the CCoAOMT gene family. Real-time PCR analysis revealed that the CcCCoAOMT1 gene is constitutively expressed in all tissues, and the expression level was greatest in stem, followed by stem bark, roots and leaves. In order to understand this gene's function, we transformed it into Arabidopsis thaliana; integration (one insertion site) was confirmed following PCR and southern hybridization. The over-expression of CcCCoAOMT1 in these transgenic A. thaliana plants resulted in increased plant height and silique length relative to non-transgenic plants. Perhaps the most important finding was that the transgenic Arabidopsis plants contained more lignin (20.44–21.26%) than did control plants (17.56%), clearly suggesting an important role of CcCCoAOMT1 gene in lignin biosynthesis. These data are important for the success of efforts to reduce jute lignin content (thereby increasing fiber quality) via CcCCoAOMT1 gene inhibition. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Lignin is the one of the primary components of plant cell walls (second only to cellulose) and is one of the most abundant biopolymers on earth. Lignin appears in the cell wall during secondary thickening, via polymerization of the monomers coumaric alcohol, coniferyl alcohol and sinapyl alcohol catalyzed by oxidative enzymes (Baucher et al., 1998). Lignin has an important role in cell wall structural integrity, stem strength, water transport, and pathogen resistance (Ruben et al., 2010; Tronchet et al., 2010; Whetten and Sederoff, 1995). However, the presence of lignin also negatively affects fiber quality. For example, in ramie, or jute fiber, the presence of lignin negatively affects degumming technologies, and subsequent textile quality (Bellaloui et al., 2012; Tian et al., 2013). Lignin also affects the digestibility of forage plants, reducing the nutritional value of that forage; so that lignin content is used as an index of forage quality (Sederoff, 1999). It is therefore important to understand the regulatory factors involved in plant lignin biosynthesis; doing so may further enhance our ability to cultivate low-lignin containing plants. Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is a key enzyme in Lignin biosynthesis process in plants. This enzyme is a type of Abbreviations: CCoAOMT, Caffeoyl-CoA O-methyltransferase. ⁎ Corresponding author. E-mail address: [email protected] (J. Qi).
S-adenosyl-L-methionine (SAM) methyltransferase that uses coffee acyl coenzyme A as its substrate. The S-adenosyl methionine on the methyl group transfers to the lignin monomer's benzene carbon 3 position, forming ferulic acyl coenzyme A, which is a key methyl transferase in lignin biosynthetic pathway (Wout et al., 2003). There are a total of eight (A, B, C, D, E, F, G and H) conserved sequence elements in the amino acid sequence of CCoAOMT gene, the A, B, and C elements are unique to plant methylases, while the D, E, F, G, and H elements are the tag sequences specific to the CCoAOMT gene family (Joshi and Chang, 1998). The CCOAOMT gene has been cloned from many plants, including Arabidopsis thaliana (Do et al., 2007; Li et al., 2010; Zhao et al., 2004), Oryza sativa (Li et al., 2008), Bambusoideae (Maury et al., 1999), Nicotiana tabacum, Zea mays (Civardi et al., 1999), and Populus alba (Suzuki et al., 2006). The first confirmation that CCoAOMT gene was involved in lignin synthesis occurred in Zinnia elegans (Meyermans et al., 2000; Zhong et al., 1998). Antisense RNA technology has been successfully used in altering in lignin content and composition in A. thaliana, N. tabacum, Z. elegans, P. alba, Pinus etc. For example, inhibition of CCoAOMT activity in transgenic tobacco significantly reduced lignin content in that species (Zhao et al., 2002; Zhong et al., 1998, 2000). In this study we isolated the CCoAOMT gene from jute (cv. Huangma 179; the primary cultivated variety in China), and inserted it into the pCAMBIA1301 expression vector. The Agrobacterium infection method (Clough and Bentm, 1998) was subsequently used to generate to
http://dx.doi.org/10.1016/j.gene.2014.05.011 0378-1119/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Zhang, G., et al., The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.011
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G. Zhang et al. / Gene xxx (2014) xxx–xxx
transgenic A. thaliana plants. Our results showed that lignin content increased in varying degrees in these transgenic lines, up to a maximum of 21.26%. Increased plant height and silique length were also observed in these transgenic plants. These data suggest that inhibition of CcCCoAOMT gene expression is indeed a viable method for reducing lignin content in jute; therefore laying the foundation for use of genetic engineering to improve fiber quality.
2.3. Sequence and phylogenetic analysis DNA was sequenced by BGI (Shanghai, China), and analyzed using the DNAclub and DNAMAN software platforms. Alignment of cDNA, and amino acids was conducted using Blast (http://blast.ncbi.nlm.nih. gov/Blast.cgi). The MEGA 4 software platform was used for phylogenetic analysis of the protein sequence encoding by CcCCoAOMT gene with that in other plants.
2. Materials and methods 2.1. Plant material and treatments Jute seeds (cv. Huangma 179) were provided by the Genetic Improvement Laboratory of the Fujian Agriculture and Forestry University. Selected plump seeds were planted in field soil amended with composted manure, and grown in a growth chamber at 14 h/10 h, 160 μmol/m2/s−1, and day/night temperatures of 34 °C/26 °C. A. thaliana, (ecotype Columbia), was provided by the Rice Research Institute, FuJian Academy of Agriculture Sciences. Intact seeds were selected and subjected to stratification for 2 days at 4 °C. All seeds were grown in prepared soil at 18 h light/6 h dark, 24 and 18 °C, and 70% RH. Flowers of fully developed plants were used as transformation receptors. 2.2. The cDNA clone of the CcCCoAOMT1 gene Total RNA of jute plants was extracted using the OMEGA isolation kit (R6827-01, USA). First strand cDNA was synthesized using the Reverse Transcription Kit (Primescript™ RT DRR037S TAKARA, China). Degenerate primers were designed according to the CCoAOMT gene sequence of other plants (Supplementary materials) as follows: sense primer: CF: 5′-GGACAACTACATVAACTACCAC-3′ and anti-sense primer: CR: 5′CTGTSACRTCTCTGAGCTCCTTCAT-3′. These degenerate primers were then used to amplify the key sequence of the CCoAOMT gene in jute, and subsequently used to design the following two primers using 5′RACE PCR: Cc-R1: 5′-TAATGTAGTT GTCCTTGTCAGC-3′ and Cc-R2: 5′-AGTTAATGTAGTTGTCCTTGTCAGC3′. cDNA (1 μL) was used as the PCR template for the first time PCR in a reaction mix containing 1 μL upstream and downstream primers (10 μmol L−1), 2.5 μL 10× buffer, 2 μL dNTPs (10 mM each), 0.2 μL polymerase (0.2 μL, 10 U/μL), brought to 25 μL with ddH2O. PCR conditions were as follows: denaturation at 94 °C for 30 s, annealing at 60 °C for 1 min, and extension at 72 °C for 2 min, for 35 cycles, the final extension was carried out at 72 °C for 10 min. The resulting PCR (1 μL) product was used as the template for the second PCR reaction, using identical reaction volumes and conditions. This final PCR product was separated on 1% agarose gel. The sequence was sub-cloned into the pUC19 vector following a standard cloning method (Clough and Bentm, 1998). The sequences were sequenced and results used in a subsequent homology search in NCBI (www.ncbi.nlm.nih.gov/). 3′RACE primers (Cc3-F1: 5′-TTGAAGCTGGGACATACCATGGAAC-3′, Cc3-F2: 5′-TGTTGATGCTGACAAGGACAACTAC-3′), PCR reaction system and the conditions were the same as used in 5′RACE. The DNAMAN software was used to assemble the 5′RACE and 3′RACE results into the full-length cDNA of CcCCoAOMT1 gene. The specific primers for the amplified full-length cDNA were designed (CcQF: 5′-GGAATTCTTGAGACCA GTGTTTAT-3′; CcQR: 5′-GCGTCGACGTAGCCGATGACTCCCCCG-3′). The PCR reaction system contained 1 μL cDNA, 1 μL upstream and downstream primers (10 μmol L− 1), 2.5 μL 10 × buffer, 2 μL dNTP, 0.2 μL Taq polymerase (10 U/μL), and brought to 25 μL with ddH2O. The PCR conditions were as follows: denaturation at 94 °C for 30 s, annealing at 58 °C for 40 s, and extension at 72 °C for 2 min for 35 cycles, the final extension was carried out at 72 °C for 10 min. The product was recycled on a 1% agarose gel. The sequences were sub-cloned into the pUC19 vector following the standard cloning method and characterized by sequencing and subsequent homology search in NCBI.
2.4. Real-time PCR analysis Total RNA was isolated from jute tissues (roots, stem bark, stem, and leaves) and cDNA was generated using a PrimeScript™ reverse transcription kit (TaKaRa, China) according to manufacturer's instructions. Resulting cDNA was diluted 10× for use as the template for expression analyses. Primers for expression analysis were designed according to the full-length cDNA sequence of the CCoAOMT gene (CPF: 5′-GAGC CAGAGCCAATGAAGGA-3′, CPR: 5′-GTCAAGAACAGGCAAAGCAG-3′) and the jute 18sRNA gene, which served as the internal reference (18s-F: 5′-GTGGAGCGATTTGTCTGGTT-3′, 18s-R: 5′-TGTACAAAGGGC AGGGACGT-3′). Expression analyses were conducted using the ABI 7300 real-time PCR system with a reaction system containing upstream and downstream primers (0.5 μL, 10 μmol L−1), 2× SYBR Green Master Mix (1 μL), cDNA (1 μL) and brought up to 20 μL with ddH2O. Quantitative real-time PCR analysis was performed using the following reaction conditions: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 50 s (40 cycles) using an ABI 7500 fluorescence quantitative PCR instrument. All experiments used three different biological samples and relative gene expression was calculated using the 2−ΔΔCT method. All data was analyzed using SPSS18.0 variance analysis. 2.5. Plasmid construction The open reading Frame of CcCCOAOMT1 gene was amplified with the sense primer (5′-GAAGATCTTTGAGACCAGTGTTTAT-3′), and antisense primer (5′-GGGTAACCGTAGCCGATGACTCCCCCG-3′) (restriction endonuclease BglII and BstEII sites are noted in underlined letters). The amplified PCR product was digested with BglII and BSTEII, and inserted into the pCAMBIA1301 binary vector containing the hygromycin phosphotransferase (hph) gene, under the control of a CaMV35S promoter. Sequence analysis confirmed proper forward insertion of CcCCOAOMT1 gene into the vector. 2.6. Plant transformation of Arabidopsis and screening The resulting vector was introduced into the Agrobacterium tumefaciens strain EHA105 by the freeze–thaw method (Holsters et al., 1978). A. thaliana plants were subsequently transformed by the floral dip method as described by Clough and Bentm (1998). Transgenic lines were obtained following selection on MS (Murashige and Skoog, 1962) culture medium supplemented with hygromycin (30 mg/L). Transgenic plants were confirmed via PCR using the gene-specific primers: 1304-F (5′-CGGGATCCATACTTGAGACCAGTGTTT-3′) and vector-specific primers: 1304-R (5′-GGGGTACCCGACCTTAACTAGCTC AAT-3′). The PCR cycle was as follows: 94 °C denaturation for 5 min, 94 °C for 50 s, 55 °C for 50 s, 72 °C for 1.5 min, for 35 cycles, extension at 72 °C for 10 min at 4 °C, and reaction termination. The PCR product was detected in 1% agarose gel electrophoresis. PCR (positive) plants were confirmed by southern blot detection. Briefly, 5–10 μg genomic DNA was digested overnight with BSTEII, BGIII and NcoI, and hybridized to a hygromycin probe labeled with alkaline phosphatase Labeling Kit (ROCHE Company). The hygromycin probe primers were as follows: R: 5′-CATACTTGAGACCAGTGTTT-3′, and F: 5′-CCGACCTTAACTAGCTCA AT-3′. Results were recorded using the gel imaging system FluorChem SP with a 50 mm f11.4 lens.
Please cite this article as: Zhang, G., et al., The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.011
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2.7. Phenotype analysis of transgenic A. thaliana Plant height and silique length of transgenic and non-transgenic plants (5 each, 3 replications) were recorded and analyzed using SPSS18.0 variance analysis. 2.8. Determination of lignin content Lignin content was determined using the micro-Klason method (Huntley et al., 2003). Briefly 100 mg of dried 60-day-old transgenic and wild A. thaliana stems were ground to pass through a 40-mesh screen, and extracted with acetone in the Soxhlet apparatus for 6 h. The acetone extraction (100 mg) was treated with 5 mL 72% H2SO4 for 2 h at 25, mixed with 112 mL distilled H2O, and steamed for 1 h. Mixtures were filtered again through a 40 mesh screen rinsed with 100 °C water, dried and weighed. The percentage of lignin was determined by dividing the remainder (sediment) by the total weight, and multiplied by 100. All data was recorded and analyzed using SPSS18.0 variance analysis.
to P. trichocarpa, yet distantly related to A. majus, C. lanceolata and S. tuberosum.
3. Results
3.3. Real-time PCR analysis of CcCCOAOMT1 expression
3.1. RNA extraction from stem bark of jute
Fluorescent quantitative PCR analysis showed that the CcCCOAOMT1 gene is expressed in root, stem bark, stem and leaf tissues, with apparent higher expression in stem (Fig. 4). The absence of significant differences between expression levels in each of these tissues suggests that the CcCCOAOMT1 gene is constitutively expressed in all tissues. Its greater expression in stem may indicate a role in plant growth, development and lodging resistance. It is likely that a higher lignin content helps plants better tolerate adverse external environments (Tronchet et al., 2010; Wei and Song, 2001).
Jute stem bark RNA was extracted using the optimized OMEGA RNA extraction kit, and integrity of total RNA was determined using 0.8% agarose gel electrophoresis. The electrophoresis pattern included three bands (28S, 18S, 5S) (Fig. 1), and the brightness of the 28S bands was 2× that of 18S. Almost no RNA degradation, or protein contamination occurred.
Fig. 2. Agarose gel electropherogram of CcCCOAOMT gene. a: RACE PCR products. M: Marker DL2000. 1, 2 and 3: 5′RACE PCR products. 4, 5 and 6: 3′RACE PCR products. 7: positive control. b (1, 2 and 3): the products of full-length cDNA. 4: positive control.
3.2. Cloning and analysis the cDNA of CcCCOAOMT1 gene After PCR amplification we obtained the key fragment of CcCCoAOMT1 gene (about 360 bp). Through 5′RACE PCR and 3′ RACE PCR amplification we obtained 300 bp and 500 bp sequences (Fig. 2a), and assembled the 360 bp, 300 bp and 500 bp sequences into a fulllength sequence. Using specific primers based on the full-length sequence, we amplificated the full-length open coding frame. We subsequently obtained a fragment about 650 bp (Fig. 2b). Sequencing verified that this gene belongs to the CcCCoAOMT gene family. Sequence assembly resulted in a full-length cDNA of 987 bp, with an open reading frame of 609 bp, which encoded 202 amino acids. After analyzing deduced amino acid sequence of CcCCoAOMT1 by BLAST in NCBI, we found the CcCCoAOMT1 had high identity with Gossypium hirsutum (FJ415165.1) (90%), Populus tremula (DQ302093.1) (84%), Prunus persica (KC339525.1) (83%), Eucalyptus grandis (EU737107.1) (82%), Pyrus pyrifolia (AB013353.1) (82%), and Vitis vinifera (FQ387683.1) (80%). A phylogenetic tree (Fig. 3) was constructed based on the amino acid sequences of the CcCCoAOMT1 proteins of Ammi majus, Codonopsis lanceolata, Medicago truncatula, Leucaena leucocephala, Solanum tuberosum, Carthamus tinctorius, Z. elegans, L. leucocephala, V. vinifera, A. thaliana, Populus trichocarpa and G. hirsutum. From these analyses it was apparent that the CcCCOAOMT1 gene is closely related
Fig. 1. Agarose gel electropherogram of total RNA from stem bark of jute. 1, 2 and 3: represent the different samples.
3.4. The PCR and southern blot analysis All the hygromycin screening positive plants contain objective fragments (Fig. 5) after PCR amplification. The result of southern hybridization (Fig. 6) shows that: the transgenic plants have one or two copies, no hybridization bands were detected in the non-transgene plants, our
Fig. 3. Phylogenetic tree of CCOAOMT1 from jute based on the full amino acid sequences. The tree was constructed by the neighbor-joining method. Amino acid sequence of CCOAOMT comes from Ammi majus (AAT40111.1), Codonopsis lanceolata (BAE48788.1), Medicago truncatula (XP_003607927.1), Leucaena leucocephala (ABE60812.1), Solanum tuberosum (BAC23054.1), Carthamus tinctorius (BAG71889.1), Zinnia elegans (AAA59389.1), Leucaena leucocephala (ABF74684.1), Vitis vinifera (NP_001268047.1), Arabidopsis thaliana (AAM66108.1), Populus trichocarpa (ABK94003.1) and Gossypium hirsutum (ACQ59095.1).
Please cite this article as: Zhang, G., et al., The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.011
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expression level
3 2.5 2 1.5 1 0.5 0
stem bark
stem
root
leaf
Fig. 4. Relative expression of CcCCOAOMT1 gene in different tissues. All the Real-time PCR data were analyzed by SPSS18.0 LSD variance analysis.
result indicates that exogenous gene was integrated into A. thaliana genome. 3.5. Phenotype observation and lignin content analysis of transgenic lines Transgenic A. thaliana plants were taller (Fig. 5a) with longer silique (Fig. 5b) relative to non-transgenic plants (Table 1). The lignin content of transgenic plants (20.44–21.26%) was significantly greater relative to that observed in non-transgenic plants (17.56%) (Table 1). 4. Discussion Jute (Corchorus capsularis L.) is one of the most important crops used for fiber production. Since jute leaf tissues are rich in secondary metabolites (e.g. polysaccharides, polyphenols) that are released following rupture of cells during grinding, these substances will interact with RNA. In addition, RNA degrading enzymes are very stable in cells thus making it difficult to isolate high quality RNA (Birtic and Krammer, 2006). In this study we successfully extract high quality RNA from jute leaves (Fig. 1). There is almost no RNA degradation, or protein contamination and it is critical for next experiment. To ascertain the expression of CcCCoAOMT1 gene in transgenic seedlings, total RNA was isolated and RT-PCR was performed using primers specific for CCoAOMT transcripts. The PCR-positive plants were selected as putative transgenic Arabidopsis. Genomic DNA was isolated from Arabidopsis leaves of three randomly selected PCR-positive plants and transformation was confirmed using Southern blot analysis with hygromycin specific probes. The results indicated that all selected plants had T-DNA integrated in Arabidopsis genome and contained one insertion site (Fig. 7). No hybridization bands were detected in the non-transgene plants. We isolated a new CcCCoAOMT1 gene from jute, and multiple sequence analyses indicated that this gene has all the characteristic elements of the CCoAOMT gene. For example the A, B, and C elements are widely conserved in plant methyltransferase enzyme sequences. And the D, E, F, G, and H tag conserved element sequences of the CCoAOMT gene family, which indicate that CcCCoAOMT1 is a member of CCoAOMT gene family. Real-time PCR indicated that the CcCCoAOMT1 gene is constitutively expressed in all tissues yet was apparently higher in stems.
Fig. 6. The result of Southern hybridization. 1: Positive control of Pcambia1301 vector. 2: Digested by BSTEII enzyme. 3: Digested by BGIII enzyme. 4: Digested by NcoI enzyme. 5: The negative control.
The function of lignified tissue (wood) in maintaining stem strength is consistent with the function of CCoAOMT gene. Similar expression patterns have been reported in other species (Zhao et al., 2004). The insertion of the CcCCoAOMT1 gene in A. thaliana resulted in increased lignin content, suggesting that CcCCoAOMT1 gene is involved in lignin biosynthesis in plants. The results of this study are similar to that found in Lilium Oriental Hybrids and Pinus radiata (Armin et al., 2011; Li, 2009). The poor quality of fiber obtained from jute is mainly due to the relatively high lignin content (much higher than that in ramie or cotton) (Lan and Zhang, 2009; Su and Gong, 2005). So, reduce the lignin content is a new way to improve the quality of the jute. Some studies have provided new insight for reducing the lignin content in the jute focusing on the CcCCoAOMT1 gene. At present there are many reports based on antisense RNA technology to reduce the content of the lignin. For example, CCoAOMT suppression modifies lignin composition in P. radiata (Armin et al., 2011; Kawaoka et al., 2006; Zhong et al., 1998). The lignin content was reduced in tobacco via suppression of two O-methyltransferases of caffeic acid O-methyltransferase (COMT) and caffeoylCoA-3-O-methyltransferase (CCoAOMT) genes (Tronchet et al., 2010; Vanholme et al., 2008; Zhao et al., 2002). CCoAOMT suppression in N. tabacum, A. thaliana, Medicago sativa and Populus resulted in lignin reductions of 20–45% (Do et al., 2007; Meyermans et al., 2000; Zhong et al., 2000). In summary, we isolated the full-length cDNA of the CCoAOMT1 gene from jute, using homology clone (Primers were designed according to the sequence of CCoAOMT gene of other species.), and a modified RACE technique. Bioinformatic analyses showed that the gene is a member of the CCoAOMT gene family. Real-time PCR analysis indicated that this gene is constitutively expressed in all tissues. PCR and southern hybridization results indicate that the exogenous gene was integrated into the Arabidopsis genome, with only one or two insertion sites. Overexpression of the CcCCoAOMT1 gene in Arabidopsis resulted in significantly taller plants, and increased silique length compared to nontransgenic plants. Perhaps most important is that transgenic Arabidopsis plants had greater lignin content compared to the non-transgenic plants. Our results suggest that the CcCCoAOMT1 gene plays a key role in lignin biosynthesis and other growth process.
Conflicts of interest All authors read and approved the final manuscript. None of the authors had any conflicts of interest. Table 1 Morphology analysis of wild-type and transgenic Arabidopsis.
Fig. 5. The result of PCR amplification. M: DL2000 Marker. 1, 2, 3, 4, 5: Represent different samples. 6: Represents negative control.
Lines
Length of silique (cm)
Height (cm)
Lignin (%)
Control Transgene line 1 Transgene line 2 Transgene line 3
1.40 1.62 1.6 1.62
34.84 37.92 38.44.0 38.66
17.56 20.44 21.26 21.06
± ± ± ±
0.14 0.14a 0.14a 0.08a
± ± ± ±
0.88 0.87a 0.77a 0.71a
± ± ± ±
0.57 0.55a 0.50a 0.57a
Each value represents mean of five replicates ± SD. Means were compared using ANOVA. Letter a after data within a column represents significant difference at 5% probability level.
Please cite this article as: Zhang, G., et al., The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.011
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Fig. 7. Morphology of wild-type and transgenic Arabidopsis. a: Transgenic Arabidopsis are higher than wild-type. b: Transgenic Arabidopsis have longer silique than wild-type.
Acknowledgments Financial support is from the Chinese Southeast of jute and kenaf Experiment Station construction project of the Ministry of Agriculture(2011.9), innovation of jute and kenaf germplasm resources sharing platform project and Natural Science Foundation of Fujian Province (2011J05046) National agricultural industry technology system (CARS-19-E06), Study on Exploration and Innovation of Germplasm in Bast fiber, Cane, Tea and Mulberry (2013BAD01B03-13), The projects of international advanced agricultural science and technology (013270). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.05.011. References Armin, Wagner, Yuki, Tobimatsu, Lorelle, Phillips, Heather, Flint, Kirk, Torr, Lloyd, Donaldson, Lana, Pears, John, Ralph, 2011. CCoAOMT suppression modifies lignin composition in Pinus radiate. Plant Journal 67, 78–96. Baucher, M., Monties, B., Van Montagu, M., Boerjanm, W., 1998. Biosynthesis and genetic engineering of lignin. Critical Reviews in Plant Sciences 17, 125–197. Bellaloui, N., Mengistu, A., Zobiole, L.H.S., Shier, W.T., 2012. Resistance to toxin-mediated fungal infection: role of lignins, isoflavones, other seed phenolics, sugars, and boron in the mechanism of resistance to charcoal rot disease in soybean. Toxin Reviews 31, 16–26. Birtic, S., Krammer, I., 2006. Isolation of high-quality RNA from polyphenol, polysaccharide and lipid rich seeds. Phytochemical Analysis 17, 8–14. Civardi, L., Rigau, J., Puigdomenech, P., 1999. Nucleotide sequence of two cDNAs coding for caffeoy-lcoenzyme A O-methyltransferase (CCoAOMT) and study of their expression in Zea mays. Plant Physiology 120, 1206–1211.
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Cinnamyl alcohol dehydrogenases C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis. Molecular Plant Pathology 11, 83–92. Vanholme, R., Morreel, K., Ralph, J., Boerjan, W., 2008. Lignin engineering. Current Opinion in Plant Biology 11, 278–285. Wei, jian. Hua, Song, yan. ru, 2001. Recent advances in student of lignin biosynthesis and manipulation. Acta Botanica Sinica 43, 771–779. Whetten, R., Sederoff, R., 1995. Lignin biosynthesis. The Plant Cell 7, 1001–1013. Wout, B., Joh, N.R., Marie, B., 2003. Lignin biosynthesis. Annual Review of Plant Biology 54, 519–546. Zhao, H.Y., Wei, J.H., Zhang, J.Y., Liu, H.R., Wang, T., Song, Y.R., 2002. Lignin biosynthesis by suppression of two O-methyltransferases. Chinese Science Bulletin 47, 1092–1095. Zhao, H.Y., Sheng, Q.X., Li, S.Y., Wang, T., Song, Y.R., 2004. Characterization of three rice CCoAOMT genes. Chinese Science Bulletin 49, 1602–1606. 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Please cite this article as: Zhang, G., et al., The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.011
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The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana Gaoyang Zhang, Yujia Zhang, Jiantang Xu, Xiaoping Niu, Jianmin Qi ⁎, Aifen Tao, Liwu Zhang, Pingping Fang, LiHui Lin, Jianguang Su Key Laboratory for Genetics, Breeding and Multiple Utilization of Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, PR China
a r t i c l e
i n f o
Article history: Received 25 September 2013 Received in revised form 27 February 2014 Accepted 3 May 2014 Available online xxxx Keywords: CcCCoAOMT RACE Jute Southern hybridization Arabidopsis
a b s t r a c t The Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is a key enzyme in lignin biosynthesis in plants. In this study we cloned the full-length cDNA of the Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) gene from jute using homology clone (primers were designed according to the sequence of CCoAOMT gene of other plants), and a modified RACE technique, subsequently named “CcCCoAOMT1”. Bioinformatic analyses showed that the gene is a member of the CCoAOMT gene family. Real-time PCR analysis revealed that the CcCCoAOMT1 gene is constitutively expressed in all tissues, and the expression level was greatest in stem, followed by stem bark, roots and leaves. In order to understand this gene's function, we transformed it into Arabidopsis thaliana; integration (one insertion site) was confirmed following PCR and southern hybridization. The over-expression of CcCCoAOMT1 in these transgenic A. thaliana plants resulted in increased plant height and silique length relative to non-transgenic plants. Perhaps the most important finding was that the transgenic Arabidopsis plants contained more lignin (20.44–21.26%) than did control plants (17.56%), clearly suggesting an important role of CcCCoAOMT1 gene in lignin biosynthesis. These data are important for the success of efforts to reduce jute lignin content (thereby increasing fiber quality) via CcCCoAOMT1 gene inhibition. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Lignin is the one of the primary components of plant cell walls (second only to cellulose) and is one of the most abundant biopolymers on earth. Lignin appears in the cell wall during secondary thickening, via polymerization of the monomers coumaric alcohol, coniferyl alcohol and sinapyl alcohol catalyzed by oxidative enzymes (Baucher et al., 1998). Lignin has an important role in cell wall structural integrity, stem strength, water transport, and pathogen resistance (Ruben et al., 2010; Tronchet et al., 2010; Whetten and Sederoff, 1995). However, the presence of lignin also negatively affects fiber quality. For example, in ramie, or jute fiber, the presence of lignin negatively affects degumming technologies, and subsequent textile quality (Bellaloui et al., 2012; Tian et al., 2013). Lignin also affects the digestibility of forage plants, reducing the nutritional value of that forage; so that lignin content is used as an index of forage quality (Sederoff, 1999). It is therefore important to understand the regulatory factors involved in plant lignin biosynthesis; doing so may further enhance our ability to cultivate low-lignin containing plants. Caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) is a key enzyme in Lignin biosynthesis process in plants. This enzyme is a type of Abbreviations: CCoAOMT, Caffeoyl-CoA O-methyltransferase. ⁎ Corresponding author. E-mail address: [email protected] (J. Qi).
S-adenosyl-L-methionine (SAM) methyltransferase that uses coffee acyl coenzyme A as its substrate. The S-adenosyl methionine on the methyl group transfers to the lignin monomer's benzene carbon 3 position, forming ferulic acyl coenzyme A, which is a key methyl transferase in lignin biosynthetic pathway (Wout et al., 2003). There are a total of eight (A, B, C, D, E, F, G and H) conserved sequence elements in the amino acid sequence of CCoAOMT gene, the A, B, and C elements are unique to plant methylases, while the D, E, F, G, and H elements are the tag sequences specific to the CCoAOMT gene family (Joshi and Chang, 1998). The CCOAOMT gene has been cloned from many plants, including Arabidopsis thaliana (Do et al., 2007; Li et al., 2010; Zhao et al., 2004), Oryza sativa (Li et al., 2008), Bambusoideae (Maury et al., 1999), Nicotiana tabacum, Zea mays (Civardi et al., 1999), and Populus alba (Suzuki et al., 2006). The first confirmation that CCoAOMT gene was involved in lignin synthesis occurred in Zinnia elegans (Meyermans et al., 2000; Zhong et al., 1998). Antisense RNA technology has been successfully used in altering in lignin content and composition in A. thaliana, N. tabacum, Z. elegans, P. alba, Pinus etc. For example, inhibition of CCoAOMT activity in transgenic tobacco significantly reduced lignin content in that species (Zhao et al., 2002; Zhong et al., 1998, 2000). In this study we isolated the CCoAOMT gene from jute (cv. Huangma 179; the primary cultivated variety in China), and inserted it into the pCAMBIA1301 expression vector. The Agrobacterium infection method (Clough and Bentm, 1998) was subsequently used to generate to
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Please cite this article as: Zhang, G., et al., The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.011
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G. Zhang et al. / Gene xxx (2014) xxx–xxx
transgenic A. thaliana plants. Our results showed that lignin content increased in varying degrees in these transgenic lines, up to a maximum of 21.26%. Increased plant height and silique length were also observed in these transgenic plants. These data suggest that inhibition of CcCCoAOMT gene expression is indeed a viable method for reducing lignin content in jute; therefore laying the foundation for use of genetic engineering to improve fiber quality.
2.3. Sequence and phylogenetic analysis DNA was sequenced by BGI (Shanghai, China), and analyzed using the DNAclub and DNAMAN software platforms. Alignment of cDNA, and amino acids was conducted using Blast (http://blast.ncbi.nlm.nih. gov/Blast.cgi). The MEGA 4 software platform was used for phylogenetic analysis of the protein sequence encoding by CcCCoAOMT gene with that in other plants.
2. Materials and methods 2.1. Plant material and treatments Jute seeds (cv. Huangma 179) were provided by the Genetic Improvement Laboratory of the Fujian Agriculture and Forestry University. Selected plump seeds were planted in field soil amended with composted manure, and grown in a growth chamber at 14 h/10 h, 160 μmol/m2/s−1, and day/night temperatures of 34 °C/26 °C. A. thaliana, (ecotype Columbia), was provided by the Rice Research Institute, FuJian Academy of Agriculture Sciences. Intact seeds were selected and subjected to stratification for 2 days at 4 °C. All seeds were grown in prepared soil at 18 h light/6 h dark, 24 and 18 °C, and 70% RH. Flowers of fully developed plants were used as transformation receptors. 2.2. The cDNA clone of the CcCCoAOMT1 gene Total RNA of jute plants was extracted using the OMEGA isolation kit (R6827-01, USA). First strand cDNA was synthesized using the Reverse Transcription Kit (Primescript™ RT DRR037S TAKARA, China). Degenerate primers were designed according to the CCoAOMT gene sequence of other plants (Supplementary materials) as follows: sense primer: CF: 5′-GGACAACTACATVAACTACCAC-3′ and anti-sense primer: CR: 5′CTGTSACRTCTCTGAGCTCCTTCAT-3′. These degenerate primers were then used to amplify the key sequence of the CCoAOMT gene in jute, and subsequently used to design the following two primers using 5′RACE PCR: Cc-R1: 5′-TAATGTAGTT GTCCTTGTCAGC-3′ and Cc-R2: 5′-AGTTAATGTAGTTGTCCTTGTCAGC3′. cDNA (1 μL) was used as the PCR template for the first time PCR in a reaction mix containing 1 μL upstream and downstream primers (10 μmol L−1), 2.5 μL 10× buffer, 2 μL dNTPs (10 mM each), 0.2 μL polymerase (0.2 μL, 10 U/μL), brought to 25 μL with ddH2O. PCR conditions were as follows: denaturation at 94 °C for 30 s, annealing at 60 °C for 1 min, and extension at 72 °C for 2 min, for 35 cycles, the final extension was carried out at 72 °C for 10 min. The resulting PCR (1 μL) product was used as the template for the second PCR reaction, using identical reaction volumes and conditions. This final PCR product was separated on 1% agarose gel. The sequence was sub-cloned into the pUC19 vector following a standard cloning method (Clough and Bentm, 1998). The sequences were sequenced and results used in a subsequent homology search in NCBI (www.ncbi.nlm.nih.gov/). 3′RACE primers (Cc3-F1: 5′-TTGAAGCTGGGACATACCATGGAAC-3′, Cc3-F2: 5′-TGTTGATGCTGACAAGGACAACTAC-3′), PCR reaction system and the conditions were the same as used in 5′RACE. The DNAMAN software was used to assemble the 5′RACE and 3′RACE results into the full-length cDNA of CcCCoAOMT1 gene. The specific primers for the amplified full-length cDNA were designed (CcQF: 5′-GGAATTCTTGAGACCA GTGTTTAT-3′; CcQR: 5′-GCGTCGACGTAGCCGATGACTCCCCCG-3′). The PCR reaction system contained 1 μL cDNA, 1 μL upstream and downstream primers (10 μmol L− 1), 2.5 μL 10 × buffer, 2 μL dNTP, 0.2 μL Taq polymerase (10 U/μL), and brought to 25 μL with ddH2O. The PCR conditions were as follows: denaturation at 94 °C for 30 s, annealing at 58 °C for 40 s, and extension at 72 °C for 2 min for 35 cycles, the final extension was carried out at 72 °C for 10 min. The product was recycled on a 1% agarose gel. The sequences were sub-cloned into the pUC19 vector following the standard cloning method and characterized by sequencing and subsequent homology search in NCBI.
2.4. Real-time PCR analysis Total RNA was isolated from jute tissues (roots, stem bark, stem, and leaves) and cDNA was generated using a PrimeScript™ reverse transcription kit (TaKaRa, China) according to manufacturer's instructions. Resulting cDNA was diluted 10× for use as the template for expression analyses. Primers for expression analysis were designed according to the full-length cDNA sequence of the CCoAOMT gene (CPF: 5′-GAGC CAGAGCCAATGAAGGA-3′, CPR: 5′-GTCAAGAACAGGCAAAGCAG-3′) and the jute 18sRNA gene, which served as the internal reference (18s-F: 5′-GTGGAGCGATTTGTCTGGTT-3′, 18s-R: 5′-TGTACAAAGGGC AGGGACGT-3′). Expression analyses were conducted using the ABI 7300 real-time PCR system with a reaction system containing upstream and downstream primers (0.5 μL, 10 μmol L−1), 2× SYBR Green Master Mix (1 μL), cDNA (1 μL) and brought up to 20 μL with ddH2O. Quantitative real-time PCR analysis was performed using the following reaction conditions: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 50 s (40 cycles) using an ABI 7500 fluorescence quantitative PCR instrument. All experiments used three different biological samples and relative gene expression was calculated using the 2−ΔΔCT method. All data was analyzed using SPSS18.0 variance analysis. 2.5. Plasmid construction The open reading Frame of CcCCOAOMT1 gene was amplified with the sense primer (5′-GAAGATCTTTGAGACCAGTGTTTAT-3′), and antisense primer (5′-GGGTAACCGTAGCCGATGACTCCCCCG-3′) (restriction endonuclease BglII and BstEII sites are noted in underlined letters). The amplified PCR product was digested with BglII and BSTEII, and inserted into the pCAMBIA1301 binary vector containing the hygromycin phosphotransferase (hph) gene, under the control of a CaMV35S promoter. Sequence analysis confirmed proper forward insertion of CcCCOAOMT1 gene into the vector. 2.6. Plant transformation of Arabidopsis and screening The resulting vector was introduced into the Agrobacterium tumefaciens strain EHA105 by the freeze–thaw method (Holsters et al., 1978). A. thaliana plants were subsequently transformed by the floral dip method as described by Clough and Bentm (1998). Transgenic lines were obtained following selection on MS (Murashige and Skoog, 1962) culture medium supplemented with hygromycin (30 mg/L). Transgenic plants were confirmed via PCR using the gene-specific primers: 1304-F (5′-CGGGATCCATACTTGAGACCAGTGTTT-3′) and vector-specific primers: 1304-R (5′-GGGGTACCCGACCTTAACTAGCTC AAT-3′). The PCR cycle was as follows: 94 °C denaturation for 5 min, 94 °C for 50 s, 55 °C for 50 s, 72 °C for 1.5 min, for 35 cycles, extension at 72 °C for 10 min at 4 °C, and reaction termination. The PCR product was detected in 1% agarose gel electrophoresis. PCR (positive) plants were confirmed by southern blot detection. Briefly, 5–10 μg genomic DNA was digested overnight with BSTEII, BGIII and NcoI, and hybridized to a hygromycin probe labeled with alkaline phosphatase Labeling Kit (ROCHE Company). The hygromycin probe primers were as follows: R: 5′-CATACTTGAGACCAGTGTTT-3′, and F: 5′-CCGACCTTAACTAGCTCA AT-3′. Results were recorded using the gel imaging system FluorChem SP with a 50 mm f11.4 lens.
Please cite this article as: Zhang, G., et al., The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.011
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2.7. Phenotype analysis of transgenic A. thaliana Plant height and silique length of transgenic and non-transgenic plants (5 each, 3 replications) were recorded and analyzed using SPSS18.0 variance analysis. 2.8. Determination of lignin content Lignin content was determined using the micro-Klason method (Huntley et al., 2003). Briefly 100 mg of dried 60-day-old transgenic and wild A. thaliana stems were ground to pass through a 40-mesh screen, and extracted with acetone in the Soxhlet apparatus for 6 h. The acetone extraction (100 mg) was treated with 5 mL 72% H2SO4 for 2 h at 25, mixed with 112 mL distilled H2O, and steamed for 1 h. Mixtures were filtered again through a 40 mesh screen rinsed with 100 °C water, dried and weighed. The percentage of lignin was determined by dividing the remainder (sediment) by the total weight, and multiplied by 100. All data was recorded and analyzed using SPSS18.0 variance analysis.
to P. trichocarpa, yet distantly related to A. majus, C. lanceolata and S. tuberosum.
3. Results
3.3. Real-time PCR analysis of CcCCOAOMT1 expression
3.1. RNA extraction from stem bark of jute
Fluorescent quantitative PCR analysis showed that the CcCCOAOMT1 gene is expressed in root, stem bark, stem and leaf tissues, with apparent higher expression in stem (Fig. 4). The absence of significant differences between expression levels in each of these tissues suggests that the CcCCOAOMT1 gene is constitutively expressed in all tissues. Its greater expression in stem may indicate a role in plant growth, development and lodging resistance. It is likely that a higher lignin content helps plants better tolerate adverse external environments (Tronchet et al., 2010; Wei and Song, 2001).
Jute stem bark RNA was extracted using the optimized OMEGA RNA extraction kit, and integrity of total RNA was determined using 0.8% agarose gel electrophoresis. The electrophoresis pattern included three bands (28S, 18S, 5S) (Fig. 1), and the brightness of the 28S bands was 2× that of 18S. Almost no RNA degradation, or protein contamination occurred.
Fig. 2. Agarose gel electropherogram of CcCCOAOMT gene. a: RACE PCR products. M: Marker DL2000. 1, 2 and 3: 5′RACE PCR products. 4, 5 and 6: 3′RACE PCR products. 7: positive control. b (1, 2 and 3): the products of full-length cDNA. 4: positive control.
3.2. Cloning and analysis the cDNA of CcCCOAOMT1 gene After PCR amplification we obtained the key fragment of CcCCoAOMT1 gene (about 360 bp). Through 5′RACE PCR and 3′ RACE PCR amplification we obtained 300 bp and 500 bp sequences (Fig. 2a), and assembled the 360 bp, 300 bp and 500 bp sequences into a fulllength sequence. Using specific primers based on the full-length sequence, we amplificated the full-length open coding frame. We subsequently obtained a fragment about 650 bp (Fig. 2b). Sequencing verified that this gene belongs to the CcCCoAOMT gene family. Sequence assembly resulted in a full-length cDNA of 987 bp, with an open reading frame of 609 bp, which encoded 202 amino acids. After analyzing deduced amino acid sequence of CcCCoAOMT1 by BLAST in NCBI, we found the CcCCoAOMT1 had high identity with Gossypium hirsutum (FJ415165.1) (90%), Populus tremula (DQ302093.1) (84%), Prunus persica (KC339525.1) (83%), Eucalyptus grandis (EU737107.1) (82%), Pyrus pyrifolia (AB013353.1) (82%), and Vitis vinifera (FQ387683.1) (80%). A phylogenetic tree (Fig. 3) was constructed based on the amino acid sequences of the CcCCoAOMT1 proteins of Ammi majus, Codonopsis lanceolata, Medicago truncatula, Leucaena leucocephala, Solanum tuberosum, Carthamus tinctorius, Z. elegans, L. leucocephala, V. vinifera, A. thaliana, Populus trichocarpa and G. hirsutum. From these analyses it was apparent that the CcCCOAOMT1 gene is closely related
Fig. 1. Agarose gel electropherogram of total RNA from stem bark of jute. 1, 2 and 3: represent the different samples.
3.4. The PCR and southern blot analysis All the hygromycin screening positive plants contain objective fragments (Fig. 5) after PCR amplification. The result of southern hybridization (Fig. 6) shows that: the transgenic plants have one or two copies, no hybridization bands were detected in the non-transgene plants, our
Fig. 3. Phylogenetic tree of CCOAOMT1 from jute based on the full amino acid sequences. The tree was constructed by the neighbor-joining method. Amino acid sequence of CCOAOMT comes from Ammi majus (AAT40111.1), Codonopsis lanceolata (BAE48788.1), Medicago truncatula (XP_003607927.1), Leucaena leucocephala (ABE60812.1), Solanum tuberosum (BAC23054.1), Carthamus tinctorius (BAG71889.1), Zinnia elegans (AAA59389.1), Leucaena leucocephala (ABF74684.1), Vitis vinifera (NP_001268047.1), Arabidopsis thaliana (AAM66108.1), Populus trichocarpa (ABK94003.1) and Gossypium hirsutum (ACQ59095.1).
Please cite this article as: Zhang, G., et al., The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.011
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expression level
3 2.5 2 1.5 1 0.5 0
stem bark
stem
root
leaf
Fig. 4. Relative expression of CcCCOAOMT1 gene in different tissues. All the Real-time PCR data were analyzed by SPSS18.0 LSD variance analysis.
result indicates that exogenous gene was integrated into A. thaliana genome. 3.5. Phenotype observation and lignin content analysis of transgenic lines Transgenic A. thaliana plants were taller (Fig. 5a) with longer silique (Fig. 5b) relative to non-transgenic plants (Table 1). The lignin content of transgenic plants (20.44–21.26%) was significantly greater relative to that observed in non-transgenic plants (17.56%) (Table 1). 4. Discussion Jute (Corchorus capsularis L.) is one of the most important crops used for fiber production. Since jute leaf tissues are rich in secondary metabolites (e.g. polysaccharides, polyphenols) that are released following rupture of cells during grinding, these substances will interact with RNA. In addition, RNA degrading enzymes are very stable in cells thus making it difficult to isolate high quality RNA (Birtic and Krammer, 2006). In this study we successfully extract high quality RNA from jute leaves (Fig. 1). There is almost no RNA degradation, or protein contamination and it is critical for next experiment. To ascertain the expression of CcCCoAOMT1 gene in transgenic seedlings, total RNA was isolated and RT-PCR was performed using primers specific for CCoAOMT transcripts. The PCR-positive plants were selected as putative transgenic Arabidopsis. Genomic DNA was isolated from Arabidopsis leaves of three randomly selected PCR-positive plants and transformation was confirmed using Southern blot analysis with hygromycin specific probes. The results indicated that all selected plants had T-DNA integrated in Arabidopsis genome and contained one insertion site (Fig. 7). No hybridization bands were detected in the non-transgene plants. We isolated a new CcCCoAOMT1 gene from jute, and multiple sequence analyses indicated that this gene has all the characteristic elements of the CCoAOMT gene. For example the A, B, and C elements are widely conserved in plant methyltransferase enzyme sequences. And the D, E, F, G, and H tag conserved element sequences of the CCoAOMT gene family, which indicate that CcCCoAOMT1 is a member of CCoAOMT gene family. Real-time PCR indicated that the CcCCoAOMT1 gene is constitutively expressed in all tissues yet was apparently higher in stems.
Fig. 6. The result of Southern hybridization. 1: Positive control of Pcambia1301 vector. 2: Digested by BSTEII enzyme. 3: Digested by BGIII enzyme. 4: Digested by NcoI enzyme. 5: The negative control.
The function of lignified tissue (wood) in maintaining stem strength is consistent with the function of CCoAOMT gene. Similar expression patterns have been reported in other species (Zhao et al., 2004). The insertion of the CcCCoAOMT1 gene in A. thaliana resulted in increased lignin content, suggesting that CcCCoAOMT1 gene is involved in lignin biosynthesis in plants. The results of this study are similar to that found in Lilium Oriental Hybrids and Pinus radiata (Armin et al., 2011; Li, 2009). The poor quality of fiber obtained from jute is mainly due to the relatively high lignin content (much higher than that in ramie or cotton) (Lan and Zhang, 2009; Su and Gong, 2005). So, reduce the lignin content is a new way to improve the quality of the jute. Some studies have provided new insight for reducing the lignin content in the jute focusing on the CcCCoAOMT1 gene. At present there are many reports based on antisense RNA technology to reduce the content of the lignin. For example, CCoAOMT suppression modifies lignin composition in P. radiata (Armin et al., 2011; Kawaoka et al., 2006; Zhong et al., 1998). The lignin content was reduced in tobacco via suppression of two O-methyltransferases of caffeic acid O-methyltransferase (COMT) and caffeoylCoA-3-O-methyltransferase (CCoAOMT) genes (Tronchet et al., 2010; Vanholme et al., 2008; Zhao et al., 2002). CCoAOMT suppression in N. tabacum, A. thaliana, Medicago sativa and Populus resulted in lignin reductions of 20–45% (Do et al., 2007; Meyermans et al., 2000; Zhong et al., 2000). In summary, we isolated the full-length cDNA of the CCoAOMT1 gene from jute, using homology clone (Primers were designed according to the sequence of CCoAOMT gene of other species.), and a modified RACE technique. Bioinformatic analyses showed that the gene is a member of the CCoAOMT gene family. Real-time PCR analysis indicated that this gene is constitutively expressed in all tissues. PCR and southern hybridization results indicate that the exogenous gene was integrated into the Arabidopsis genome, with only one or two insertion sites. Overexpression of the CcCCoAOMT1 gene in Arabidopsis resulted in significantly taller plants, and increased silique length compared to nontransgenic plants. Perhaps most important is that transgenic Arabidopsis plants had greater lignin content compared to the non-transgenic plants. Our results suggest that the CcCCoAOMT1 gene plays a key role in lignin biosynthesis and other growth process.
Conflicts of interest All authors read and approved the final manuscript. None of the authors had any conflicts of interest. Table 1 Morphology analysis of wild-type and transgenic Arabidopsis.
Fig. 5. The result of PCR amplification. M: DL2000 Marker. 1, 2, 3, 4, 5: Represent different samples. 6: Represents negative control.
Lines
Length of silique (cm)
Height (cm)
Lignin (%)
Control Transgene line 1 Transgene line 2 Transgene line 3
1.40 1.62 1.6 1.62
34.84 37.92 38.44.0 38.66
17.56 20.44 21.26 21.06
± ± ± ±
0.14 0.14a 0.14a 0.08a
± ± ± ±
0.88 0.87a 0.77a 0.71a
± ± ± ±
0.57 0.55a 0.50a 0.57a
Each value represents mean of five replicates ± SD. Means were compared using ANOVA. Letter a after data within a column represents significant difference at 5% probability level.
Please cite this article as: Zhang, G., et al., The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.011
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Fig. 7. Morphology of wild-type and transgenic Arabidopsis. a: Transgenic Arabidopsis are higher than wild-type. b: Transgenic Arabidopsis have longer silique than wild-type.
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Please cite this article as: Zhang, G., et al., The CCoAOMT1 gene from jute (Corchorus capsularis L.) is involved in lignin biosynthesis in Arabidopsis thaliana, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.05.011
