Accepted Article
Received Date : 23-May-2014 Revised Date
: 11-Aug-2014
Accepted Date : 15-Aug-2014 Article type
: Original Article
ORIGINAL RESEARCH PAPER Genetic diversity and expression profiles of cysteine phytases in the sheep rumen during a feeding cycle Zhongyuan Li#, a, b · Huoqing Huang#, a · Heng Zhao*, a · Kun Menga · Junqi Zhaoc · Pengjun Shi a · Peilong Yang a · Huiying Luo a· Yaru Wang a · Bin Yao*, a
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, Chinaa; Key Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science and Technology, Tianjin 300457, Chinab; National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, Chinac
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12318 This article is protected by copyright. All rights reserved.
Accepted Article
Running title: Cysteine phytase diversity in rumen
#
Z.L. and H.H. contributed equally to this article.
*Corresponding authors: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China. Tel.: +86-10-82106065. Fax: +86-10-82106054. e-mail: [email protected] (H. Zhao); [email protected] (B. Yao)
SIGNIFICANCE AND IMPACT OF THE STUDY
Ruminal phytases, i.e. cysteine phytases, are novel in sequences and functions. Great divergence in the constitution and abundance of cysteine phytase genes at the genome and transcriptional levels suggested that transcript data are more reliable to reflect the information of functional genes. Phylogenetic and rarefaction analyses indicated that the cysteine phytase genes from uncultured bacteria instead of Firmicutes play the major phytate-degrading role in rumen, and their constitution is dynamic at different time points. This study provides a new insight into ruminal cysteine phytase genes and undermines their expression profiles over a whole feeding cycle. This article is protected by copyright. All rights reserved.
Accepted Article
ABSTRACT
Cysteine phytase is the main phytate-degrading enzyme of ruminant animals. To explore the genetic diversity and dynamic expression profile of cysteine phytase in sheep rumen during a feeding cycle, four transcript (0, 4, 9 and 16 h after feeding) and one DNA (9 h after feeding) clone libraries were constructed, respectively. A total of 46 distinct gene fragments were identified, and most of these sequences had low identities (< 60%) with known phytases. Great divergence was found in the constitution and abundance of genes at the genome and transcriptional levels, and the transcript data are more reliable to reflect the information of functional genes. Phylogenetic analysis indicated that the genes from uncultured bacteria instead of Firmicutes played the major phytate-degrading role. Further comparative analysis revealed the dynamic constitution of cysteine phytase genes in rumen at different time points. KEYWORDS Clone library, Cysteine phytase, Rumen, Small tail han sheep, Transcriptional profile
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INTRODUCTION
Phytase (EC 3.2.3.8 and EC 3.2.3.26) can degrade phytate into inositol or lower inositol polyphosphate isomers and inorganic phosphates (Rao et al. 2009). Compared with monogastric animals that can’t utilize phytic acid and excrete it to the lumen of gastrointestinal tract, ruminants that harbor abundant phytase-producing bacteria, fungi and protozoa in the rumen could efficiently utilize the phytate phosphorus in feed and thus reduce the phosphorus pollution (Yanke et al. 1998). Until now, four distinctly different classes of phytases are recognized: histidine acid phosphatase, β-propeller phytase, cysteine phosphatase, and purple acid phytases, and cysteine phytase is the most abundant phytase in the rumen (Huang et al. 2011). Rumen microbial environment is highly dynamic and changes constantly with feeding. It has been reported that the amounts of ruminal anaerobic fungi and Fibrobacter succinogenes were increased by 3.6 folds and 5.4 folds, respectively, from 0 to 12 h after feeding, and that of Ruminococcus flavefaciens maintained more or less consistent (Denman and McSweeney 2006). Our previous study has shown that, during a 24-h feeding cycle, the bacterial population in sheep rumen fluid kept increasing, reached the maximum at 12 h, and then decreased in the next 12 h slowly (Li et al. 2013). In this changing environment, ruminal microbes are likely to produce functional enzymes with different pH preferences. For example, several
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known ruminal cysteine phytases have the maximum activity at pH 4.5 (Puhl et al. 2008) and pH 6.06.5 (Huang et al. 2011; Li et al. 2013), respectively. These enzymes play roles individually or in combination over the feeding cycle to degrade phytate. Transcriptional approach is widely used to investigate functional and authentic genes in a specific environment (Kellner et al. 2010; Qi et al. 2011). By using this method, we have studied the gene transcription of GH10 xylanase in the rumen during a whole feeding cycle, and revealed the expression profiles of functional xylanase genes at the transcriptional level (Li et al. 2013). In order to explore the gene diversity and expression profile of cysteine phytase in the rumen, we retrieved gene fragments by using a PCR-based approach at both genomic and transcriptional levels, and further compared their abundance over the course of a feeding cycle.
RESULTS AND DISCUSSION
Using the DNA (~100 ng μl–1, 9 h after feeding) and RNA (~400 ng μl–1, 0 h, 4 h, 9 h and 16 h after feeding) samples of the rumen contents of Small Tail Han sheep as the templates, gene fragments of cysteine phytase were amplified with degenerate primers CPhy-F and CPhy-R and transformed into E. coli for sequencing. After removing those incorrect clones identified by BLASTp, 641 sequences were identified to be putative cysteine phytases, respectively. The redundant sequences (cutting off > 95%) were removed by CD-hit software, and 23, 14, 23, 19 and 17 distinct This article is protected by copyright. All rights reserved.
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sequences were identified in the 0, 4, 9, 16 h cDNA and 9 h DNA clone libraries, respectively. These putative cysteine phytases shared 40–96% identity to known cysteine phytases, and 86% of them shared 61% relative abundances, and three genes scp1, scp2 and scp3 exist in all four cDNA clone libraries. The results indicate that during the whole feeding cycle, phytases from different rumen microbes play the major role in phytate degradation. Under harsh condition at 4 h, scp1 is distinguished in high relative abundance (79.43%) and represents the main phytate-degrader at this time point. When the rumen system becomes favorable, other genes like scp2, scp3, scp5 and scp9 also play a major role in phytate degradation. Of the 9 h DNA and cDNA clone libraries, twelve identical genes with different abundance were observed in both libraries (above 95% identity). Of the 17 distinct genes of 9 h DNA clone library, scp5 This article is protected by copyright. All rights reserved.
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and scp2 were the most predominant genes, accounting for 66% of the relative abundance, and other 15 genes had the average relative abundance of approximately 2%. In the cDNA clone library of the same time point, scp2 and scp3 were found to be the most abundant transcribed gene (61%), and other 13 genes had the average relative abundance of less than 3%. The results indicate that although there are many phytase-producing microbes in rumen, not all of them secrete functional phytases at some time point. Those genes predominant in DNA library were not highly transcribed to degrade phytate. Therefore, transcriptional approach is more reliable and efficient to study the functional phytase genes in rumen ecosystem.
MATERIALS AND METHODS
Collection of rumen contents All animal experimentation was conducted following the regulation for the review committee of laboratory animal welfare and ethics, Beijing Administration Office of Laboratory Animal, China. Three Small Tail Han sheep with cannula were fed with 10% barley, 18% wheat bran, 60% corn stalk and 12% soybean meal once a day (at 8:00 am) for one month. On the sampling day, rumen contents (~50 g) were collected prior to feeding (at 0 h) and then at 2, 4, 6, 9, 12, 16, 20 and 24 h after feeding. The rumen samples were immediately centrifuged at 16,000 × g at 4 °C for This article is protected by copyright. All rights reserved.
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10 min. The precipitates of rumen content were divided into two parts, one incubated with RNAlater reagent (Qiagen) and stored at –80 °C for RNA extraction, and the other immediately stored at –80 °C for DNA extraction.
DNA and RNA extraction Microbial DNA of each rumen content sample was extracted using a modified sodium dodecyl sulfate (SDS)-based method. The genomic DNA was further purified by an Agarose Gel DNA Purification kit (Omega) for DNA clone library construction. The rumen contents mixed with RNAlater were firstly centrifuged to remove the liquid and then ground into a fine powder in liquid nitrogen. Total RNA was extracted using the SV Total RNA Isolation System kit (Promega). The quantity and purity of RNA was measured using an Ultrospec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) based on the absorbance ratios of A260/A280 and A260/A230. Approximately 1 µg of total RNA was added in the transcript system to synthesize the first-strand cDNA using an iScript cDNA synthesis kit (Bio-Rad). These cDNAs were used for construction of the cDNA clone libraries.
Construction of five clone libraries Based on our previous study that pHs vary a lot with time after feeding (Li et al. 2013), the cDNA samples of four time points (0, 4, 9 and 16 h after feeding) were
This article is protected by copyright. All rights reserved.
Accepted Article
selected as templates for the construction of cDNA clone libraries, and the genomic DNA of the rumen content at 9 h after feeding was used to construct a DNA clone library. All libraries were prepared by degenerate PCR. The PCR was amplificated containing 1 µg of genomic DNA or cDNA as template and 10 µmol l1 of each degenerate primer CPhy-F and CPhy-R at responding PCR conditions (Huang et al. 2011). The products with correct sizes (380–400 bp) were purified, ligated with pGEM-T easy vector (Promega) and transformed into Escherichia coli TransI-T1 competent cells by heat shock (TransGen). The cells were grown at 37 °C overnight on Luria-Bertani plates, The positive white clones were verified by PCR amplification with primers M13F (5'-GTAAAACGACGGCCAGT-3') and M13R (5'-GGATAACAATTTCACACAGGA-3') and sequencing by Biomed.
Sequence analysis The partial cysteine phytase gene sequences were translated into correct reading frames by the online tool EMBOSS Transeq (http://www.ebi.ac.uk/emboss/transeq). The sequences were aligned by the BLASTp (www.ncbi.nlm.nih.gov/BLAST/) to identify putative cysteine phytases, and those correct sequences were analyzed by the CD-hit software to delete the redundant sequences (identity above 95%) (Li and Godzik 2006). To analyze the homology of these cysteine phytase sequences, seven representative cysteine phytases from the NCBI database were used as references. The deduced amino acid sequences of the cysteine phytase gene fragments and the
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reference genes were aligned by ClustalW. A phylogenetic tree was constructed using the Neighbor-Joining method with MEGA 4.0 (Tamura et al. 2007). The tree topology confidence was evaluated by the bootstrap value obtained from 1,000 replicates.
Abundance analysis In order to investigate the abundance and diversity of cysteine phytase genes during the feeding cycle, their deduced amino acid sequences were analyzed by PHYLIP (http://evolution.genetics.washington.edu/phylip.html). Rarefraction curve was then generated at the level of 6%, which was calculated by the distance-based operational taxonomic unit (OTU) and richness determination software DOTUR (Schloss and Handelsman 2005).
Nucleotide sequence accession numbers The nucleotide sequences of the cysteine phytase gene fragments were deposited into GenBank database under accession numbers KJ584400–KJ584445.
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ACKNOWLEDGEMENTS
This work was supported by the Natural Science Foundation of China (31370044) and the National Science and Technology Support Program (2011BADB02) and the China Modern Agriculture Research System (CARS-42).
CONFLICT OF INTEREST
No conflict of interest declared.
REFERENCES Denman, S.E. and McSweeney, C.S. (2006) Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS Microbiol Ecol 58, 572582. Huang, H., Zhang, R., Fu, D., Luo, J., Li, Z., Luo, H., Shi, P., Yang, P., Diao, Q. and Yao, B. (2011) Diversity, abundance and characterization of ruminal cysteine phytases suggest their important role in phytate degradation. Environ Microbiol 13, 747757.
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Jami, E. and Mizrahi, I. (2012) Composition and similarity of bovine rumen microbiota across individual animals. PLoS ONE 7, e33306. Kellner, H., Zak, D.R. and Vandenbol, M. (2010) Fungi unearthed: transcripts encoding lignocellulolytic and chitinolytic enzymes in forest soil. PLoS ONE 5, e10971. Li, W. and Godzik, A. (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22, 16581659. Li, Z., Zhao, H., Yang, P., Zhao, J., Huang, H., Xue, X., Zhang, X., Diao, Q. and Yao, B. (2013) Comparative quantitative analysis of gene expression profiles of glycoside hydrolase family 10 xylanases in the sheep rumen during a feeding cycle. Appl Environ Microbiol 79, 12121220. Puhl, A.A., Greiner, R. and Selinger, L.B. (2008) A protein tyrosine phosphatase-like inositol polyphosphatase from Selenomonas ruminantium subsp. lactilytica has specificity for the 5-phosphate of myo-inositol hexakisphosphate. Int J Biochem Cell B 40, 20532064. Qi, M., Wang, P., O'Toole, N., Barboza, P.S., Ungerfeld, E., Leigh, M.B., Selinger, L.B., Butler, G., Tsang, A. and McAllister, T.A. (2011) Snapshot of the eukaryotic gene expression in muskoxen rumen—a metatranscriptomic approach. PLoS ONE 6, e20521.
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Rao, D., Rao, K., Reddy, T. and Reddy, V. (2009) Molecular characterization, physicochemical properties, known and potential applications of phytases: an overview. Crit Rev Biotechnol 29, 182198. Schloss, P.D. and Handelsman, J. (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol 71, 15011506. Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 15961599. Wang, G., Luo, H., Meng, K., Wang, Y., Huang, H., Shi, P., Pan, X., Yang, P., Diao, Q., Zhang, H. and Yao, B. (2011) High genetic diversity and different distributions of glycosyl hydrolase family 10 and 11 xylanases in the goat rumen. PLoS ONE 6, e16731. Yanke, L., Bae, H., Selinger, L. and Cheng, K. (1998) Phytase activity of anaerobic ruminal bacteria. Microbiology 144, 15651573. Yuan, P., Meng, K., Wang, Y., Luo, H., Huang, H., Shi, P., Bai, Y., Yang, P. and Yao, B. (2012) Abundance and genetic diversity of microbial polygalacturonase and pectate lyase in the sheep rumen ecosystem. PLoS ONE 7, e40940.
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SUPPOTING INFORMATION
Fig. S1 Rarefaction curves of the cysteine phytase gene fragments from the rumen contents of Small Tail Han sheep. The gene types were defined at the 6% cut-off. The curves represent the number of gene types as a function of total clones sequenced in libraries constructed from four cDNA and one genomic DNA samples
Table 1 Amino acid sequence identity distribution of the ruminal cysteine phytase fragments retrieved from the Small Tail Han sheep
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Sequence
Percentage of cysteine phytase fragments (%)
identity (%)
cDNA of 0 h
cDNA of 4 h
cDNA of 9 h
cDNA of 16 h
DNA of 9 h
80–100
34.78
0
4.35
5.26
5.88
60–80
0
0
8.70
0
0
50–60
34.78
50.00
43.48
52.63
41.18
40–50
30.43
50.00
43.48
42.11
52.94
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Table 2 Identical cysteine phytase gene fragments from the DNA and cDNA clone libraries*
cDNA of 0 h
cDNA of 4 h
cDNA of 9 h
OTU
OT
OT
cDNA of 16 h
DNA of 9 h
Gen e
Abundan ce
scp1
0-1
scp2
0-2
scp3
0-4
scp5
0-3
U
ce
14.08
4-6
46.48
4-1
(%)
(%)
(%)
Abundanc OTU
ce
79.43
9-3
2.97
16-4 3.39
6.38
9-5
43.56
16-1 27.97
G9-5
ce (%)
e (%)
16-6
G9-9
23.74
9-2 6.34 13.38
4.26 4-4
3
17.82
0.7
1.69
7.91
16-5 5.08 9-1
0-6
0-7
Abundan
U
4-7
16-2 6.34
scp6 scp7
Abundan OTU
b
scp4
Abundan
3 4-3
4.95
2.83
1.41
G9-2 11.01
16-3 5.93 9-4
4.95
42.45 G9-3
1.44
G9-12
1.44
4-1
scp8
9-6 5
0.7
16-8 1.98
G9-7 1.69
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5.04
4-2
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scp9
0.7
scp1
9-2
6.93
16-7 33.9
G9-6
6.47
9-1 G9-10
0
4
scp1
9-1
1.05
1.44
G9-4
1
0
scp1
1.05
4-1
2.16 16-1
0-11
2
0.7
4
0.7
scp1
0
0.85
9-2 4-9
3
G9-29 0.7
scp1
1
1.05
9-2
1.44 16-1
0-17
4
0.7
0
1.05
7
0.85
scp1
0-8
5
9-7 0.7
G9-11 1.05
0.92
scp1
0-13
6
G9-13 0.7
scp1
0.92 4-1
16-1
0-18
7
0.7
scp1
2
0.7
4-1
4
0.85
16-1
0-20
8
0.7
0
0.7
1
0.85
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scp1
4-1 0-25
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9
G9-25 0.7
scp2
1
0.7
0.92
4-1 G9-16
0
6
0.7
0.92
scp2
4-5
1
9-8 0.7
G9-15 1.05
scp2
16-1
0-26
2
0.92
9-1 0.7
1.05
scp2
2
0.85
9-1 1.05
3
G9-26
6
0.92
scp2
16-9
4
Total 14
G9-19 0.85
93.63
14
100
15
91.56
14
95.76
*
The most predominant gene fragments are shown in bold.
†
OTU, operational taxonomic unit.
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0.92 17
100
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FIGURE LEGEND
Fig. 1 Phylogenetic analysis of the partial amino acid sequences of cysteine phytase retrieved from the ruminal genomic DNA and cDNA of Small Tail Han sheep and reference sequences. The microbial sources and GenBank accession numbers (in parentheses) are shown. The tree was constructed using the Neighbor-Joining method in MEGA 4. Branch lengths indicate the relative divergence among the amino acid sequences, and bootstrap values are expressed as percentages from 1,000 replications. Representative genes used for further quantitative analysis are shown in bold
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Accepted Article This article is protected by copyright. All rights reserved.
Received Date : 23-May-2014 Revised Date
: 11-Aug-2014
Accepted Date : 15-Aug-2014 Article type
: Original Article
ORIGINAL RESEARCH PAPER Genetic diversity and expression profiles of cysteine phytases in the sheep rumen during a feeding cycle Zhongyuan Li#, a, b · Huoqing Huang#, a · Heng Zhao*, a · Kun Menga · Junqi Zhaoc · Pengjun Shi a · Peilong Yang a · Huiying Luo a· Yaru Wang a · Bin Yao*, a
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, Chinaa; Key Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science and Technology, Tianjin 300457, Chinab; National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, Chinac
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12318 This article is protected by copyright. All rights reserved.
Accepted Article
Running title: Cysteine phytase diversity in rumen
#
Z.L. and H.H. contributed equally to this article.
*Corresponding authors: Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, P. R. China. Tel.: +86-10-82106065. Fax: +86-10-82106054. e-mail: [email protected] (H. Zhao); [email protected] (B. Yao)
SIGNIFICANCE AND IMPACT OF THE STUDY
Ruminal phytases, i.e. cysteine phytases, are novel in sequences and functions. Great divergence in the constitution and abundance of cysteine phytase genes at the genome and transcriptional levels suggested that transcript data are more reliable to reflect the information of functional genes. Phylogenetic and rarefaction analyses indicated that the cysteine phytase genes from uncultured bacteria instead of Firmicutes play the major phytate-degrading role in rumen, and their constitution is dynamic at different time points. This study provides a new insight into ruminal cysteine phytase genes and undermines their expression profiles over a whole feeding cycle. This article is protected by copyright. All rights reserved.
Accepted Article
ABSTRACT
Cysteine phytase is the main phytate-degrading enzyme of ruminant animals. To explore the genetic diversity and dynamic expression profile of cysteine phytase in sheep rumen during a feeding cycle, four transcript (0, 4, 9 and 16 h after feeding) and one DNA (9 h after feeding) clone libraries were constructed, respectively. A total of 46 distinct gene fragments were identified, and most of these sequences had low identities (< 60%) with known phytases. Great divergence was found in the constitution and abundance of genes at the genome and transcriptional levels, and the transcript data are more reliable to reflect the information of functional genes. Phylogenetic analysis indicated that the genes from uncultured bacteria instead of Firmicutes played the major phytate-degrading role. Further comparative analysis revealed the dynamic constitution of cysteine phytase genes in rumen at different time points. KEYWORDS Clone library, Cysteine phytase, Rumen, Small tail han sheep, Transcriptional profile
This article is protected by copyright. All rights reserved.
Accepted Article
INTRODUCTION
Phytase (EC 3.2.3.8 and EC 3.2.3.26) can degrade phytate into inositol or lower inositol polyphosphate isomers and inorganic phosphates (Rao et al. 2009). Compared with monogastric animals that can’t utilize phytic acid and excrete it to the lumen of gastrointestinal tract, ruminants that harbor abundant phytase-producing bacteria, fungi and protozoa in the rumen could efficiently utilize the phytate phosphorus in feed and thus reduce the phosphorus pollution (Yanke et al. 1998). Until now, four distinctly different classes of phytases are recognized: histidine acid phosphatase, β-propeller phytase, cysteine phosphatase, and purple acid phytases, and cysteine phytase is the most abundant phytase in the rumen (Huang et al. 2011). Rumen microbial environment is highly dynamic and changes constantly with feeding. It has been reported that the amounts of ruminal anaerobic fungi and Fibrobacter succinogenes were increased by 3.6 folds and 5.4 folds, respectively, from 0 to 12 h after feeding, and that of Ruminococcus flavefaciens maintained more or less consistent (Denman and McSweeney 2006). Our previous study has shown that, during a 24-h feeding cycle, the bacterial population in sheep rumen fluid kept increasing, reached the maximum at 12 h, and then decreased in the next 12 h slowly (Li et al. 2013). In this changing environment, ruminal microbes are likely to produce functional enzymes with different pH preferences. For example, several
This article is protected by copyright. All rights reserved.
Accepted Article
known ruminal cysteine phytases have the maximum activity at pH 4.5 (Puhl et al. 2008) and pH 6.06.5 (Huang et al. 2011; Li et al. 2013), respectively. These enzymes play roles individually or in combination over the feeding cycle to degrade phytate. Transcriptional approach is widely used to investigate functional and authentic genes in a specific environment (Kellner et al. 2010; Qi et al. 2011). By using this method, we have studied the gene transcription of GH10 xylanase in the rumen during a whole feeding cycle, and revealed the expression profiles of functional xylanase genes at the transcriptional level (Li et al. 2013). In order to explore the gene diversity and expression profile of cysteine phytase in the rumen, we retrieved gene fragments by using a PCR-based approach at both genomic and transcriptional levels, and further compared their abundance over the course of a feeding cycle.
RESULTS AND DISCUSSION
Using the DNA (~100 ng μl–1, 9 h after feeding) and RNA (~400 ng μl–1, 0 h, 4 h, 9 h and 16 h after feeding) samples of the rumen contents of Small Tail Han sheep as the templates, gene fragments of cysteine phytase were amplified with degenerate primers CPhy-F and CPhy-R and transformed into E. coli for sequencing. After removing those incorrect clones identified by BLASTp, 641 sequences were identified to be putative cysteine phytases, respectively. The redundant sequences (cutting off > 95%) were removed by CD-hit software, and 23, 14, 23, 19 and 17 distinct This article is protected by copyright. All rights reserved.
Accepted Article
sequences were identified in the 0, 4, 9, 16 h cDNA and 9 h DNA clone libraries, respectively. These putative cysteine phytases shared 40–96% identity to known cysteine phytases, and 86% of them shared 61% relative abundances, and three genes scp1, scp2 and scp3 exist in all four cDNA clone libraries. The results indicate that during the whole feeding cycle, phytases from different rumen microbes play the major role in phytate degradation. Under harsh condition at 4 h, scp1 is distinguished in high relative abundance (79.43%) and represents the main phytate-degrader at this time point. When the rumen system becomes favorable, other genes like scp2, scp3, scp5 and scp9 also play a major role in phytate degradation. Of the 9 h DNA and cDNA clone libraries, twelve identical genes with different abundance were observed in both libraries (above 95% identity). Of the 17 distinct genes of 9 h DNA clone library, scp5 This article is protected by copyright. All rights reserved.
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and scp2 were the most predominant genes, accounting for 66% of the relative abundance, and other 15 genes had the average relative abundance of approximately 2%. In the cDNA clone library of the same time point, scp2 and scp3 were found to be the most abundant transcribed gene (61%), and other 13 genes had the average relative abundance of less than 3%. The results indicate that although there are many phytase-producing microbes in rumen, not all of them secrete functional phytases at some time point. Those genes predominant in DNA library were not highly transcribed to degrade phytate. Therefore, transcriptional approach is more reliable and efficient to study the functional phytase genes in rumen ecosystem.
MATERIALS AND METHODS
Collection of rumen contents All animal experimentation was conducted following the regulation for the review committee of laboratory animal welfare and ethics, Beijing Administration Office of Laboratory Animal, China. Three Small Tail Han sheep with cannula were fed with 10% barley, 18% wheat bran, 60% corn stalk and 12% soybean meal once a day (at 8:00 am) for one month. On the sampling day, rumen contents (~50 g) were collected prior to feeding (at 0 h) and then at 2, 4, 6, 9, 12, 16, 20 and 24 h after feeding. The rumen samples were immediately centrifuged at 16,000 × g at 4 °C for This article is protected by copyright. All rights reserved.
Accepted Article
10 min. The precipitates of rumen content were divided into two parts, one incubated with RNAlater reagent (Qiagen) and stored at –80 °C for RNA extraction, and the other immediately stored at –80 °C for DNA extraction.
DNA and RNA extraction Microbial DNA of each rumen content sample was extracted using a modified sodium dodecyl sulfate (SDS)-based method. The genomic DNA was further purified by an Agarose Gel DNA Purification kit (Omega) for DNA clone library construction. The rumen contents mixed with RNAlater were firstly centrifuged to remove the liquid and then ground into a fine powder in liquid nitrogen. Total RNA was extracted using the SV Total RNA Isolation System kit (Promega). The quantity and purity of RNA was measured using an Ultrospec 2100 pro UV/visible spectrophotometer (Amersham Biosciences) based on the absorbance ratios of A260/A280 and A260/A230. Approximately 1 µg of total RNA was added in the transcript system to synthesize the first-strand cDNA using an iScript cDNA synthesis kit (Bio-Rad). These cDNAs were used for construction of the cDNA clone libraries.
Construction of five clone libraries Based on our previous study that pHs vary a lot with time after feeding (Li et al. 2013), the cDNA samples of four time points (0, 4, 9 and 16 h after feeding) were
This article is protected by copyright. All rights reserved.
Accepted Article
selected as templates for the construction of cDNA clone libraries, and the genomic DNA of the rumen content at 9 h after feeding was used to construct a DNA clone library. All libraries were prepared by degenerate PCR. The PCR was amplificated containing 1 µg of genomic DNA or cDNA as template and 10 µmol l1 of each degenerate primer CPhy-F and CPhy-R at responding PCR conditions (Huang et al. 2011). The products with correct sizes (380–400 bp) were purified, ligated with pGEM-T easy vector (Promega) and transformed into Escherichia coli TransI-T1 competent cells by heat shock (TransGen). The cells were grown at 37 °C overnight on Luria-Bertani plates, The positive white clones were verified by PCR amplification with primers M13F (5'-GTAAAACGACGGCCAGT-3') and M13R (5'-GGATAACAATTTCACACAGGA-3') and sequencing by Biomed.
Sequence analysis The partial cysteine phytase gene sequences were translated into correct reading frames by the online tool EMBOSS Transeq (http://www.ebi.ac.uk/emboss/transeq). The sequences were aligned by the BLASTp (www.ncbi.nlm.nih.gov/BLAST/) to identify putative cysteine phytases, and those correct sequences were analyzed by the CD-hit software to delete the redundant sequences (identity above 95%) (Li and Godzik 2006). To analyze the homology of these cysteine phytase sequences, seven representative cysteine phytases from the NCBI database were used as references. The deduced amino acid sequences of the cysteine phytase gene fragments and the
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reference genes were aligned by ClustalW. A phylogenetic tree was constructed using the Neighbor-Joining method with MEGA 4.0 (Tamura et al. 2007). The tree topology confidence was evaluated by the bootstrap value obtained from 1,000 replicates.
Abundance analysis In order to investigate the abundance and diversity of cysteine phytase genes during the feeding cycle, their deduced amino acid sequences were analyzed by PHYLIP (http://evolution.genetics.washington.edu/phylip.html). Rarefraction curve was then generated at the level of 6%, which was calculated by the distance-based operational taxonomic unit (OTU) and richness determination software DOTUR (Schloss and Handelsman 2005).
Nucleotide sequence accession numbers The nucleotide sequences of the cysteine phytase gene fragments were deposited into GenBank database under accession numbers KJ584400–KJ584445.
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ACKNOWLEDGEMENTS
This work was supported by the Natural Science Foundation of China (31370044) and the National Science and Technology Support Program (2011BADB02) and the China Modern Agriculture Research System (CARS-42).
CONFLICT OF INTEREST
No conflict of interest declared.
REFERENCES Denman, S.E. and McSweeney, C.S. (2006) Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen. FEMS Microbiol Ecol 58, 572582. Huang, H., Zhang, R., Fu, D., Luo, J., Li, Z., Luo, H., Shi, P., Yang, P., Diao, Q. and Yao, B. (2011) Diversity, abundance and characterization of ruminal cysteine phytases suggest their important role in phytate degradation. Environ Microbiol 13, 747757.
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Jami, E. and Mizrahi, I. (2012) Composition and similarity of bovine rumen microbiota across individual animals. PLoS ONE 7, e33306. Kellner, H., Zak, D.R. and Vandenbol, M. (2010) Fungi unearthed: transcripts encoding lignocellulolytic and chitinolytic enzymes in forest soil. PLoS ONE 5, e10971. Li, W. and Godzik, A. (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics 22, 16581659. Li, Z., Zhao, H., Yang, P., Zhao, J., Huang, H., Xue, X., Zhang, X., Diao, Q. and Yao, B. (2013) Comparative quantitative analysis of gene expression profiles of glycoside hydrolase family 10 xylanases in the sheep rumen during a feeding cycle. Appl Environ Microbiol 79, 12121220. Puhl, A.A., Greiner, R. and Selinger, L.B. (2008) A protein tyrosine phosphatase-like inositol polyphosphatase from Selenomonas ruminantium subsp. lactilytica has specificity for the 5-phosphate of myo-inositol hexakisphosphate. Int J Biochem Cell B 40, 20532064. Qi, M., Wang, P., O'Toole, N., Barboza, P.S., Ungerfeld, E., Leigh, M.B., Selinger, L.B., Butler, G., Tsang, A. and McAllister, T.A. (2011) Snapshot of the eukaryotic gene expression in muskoxen rumen—a metatranscriptomic approach. PLoS ONE 6, e20521.
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Rao, D., Rao, K., Reddy, T. and Reddy, V. (2009) Molecular characterization, physicochemical properties, known and potential applications of phytases: an overview. Crit Rev Biotechnol 29, 182198. Schloss, P.D. and Handelsman, J. (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol 71, 15011506. Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 15961599. Wang, G., Luo, H., Meng, K., Wang, Y., Huang, H., Shi, P., Pan, X., Yang, P., Diao, Q., Zhang, H. and Yao, B. (2011) High genetic diversity and different distributions of glycosyl hydrolase family 10 and 11 xylanases in the goat rumen. PLoS ONE 6, e16731. Yanke, L., Bae, H., Selinger, L. and Cheng, K. (1998) Phytase activity of anaerobic ruminal bacteria. Microbiology 144, 15651573. Yuan, P., Meng, K., Wang, Y., Luo, H., Huang, H., Shi, P., Bai, Y., Yang, P. and Yao, B. (2012) Abundance and genetic diversity of microbial polygalacturonase and pectate lyase in the sheep rumen ecosystem. PLoS ONE 7, e40940.
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SUPPOTING INFORMATION
Fig. S1 Rarefaction curves of the cysteine phytase gene fragments from the rumen contents of Small Tail Han sheep. The gene types were defined at the 6% cut-off. The curves represent the number of gene types as a function of total clones sequenced in libraries constructed from four cDNA and one genomic DNA samples
Table 1 Amino acid sequence identity distribution of the ruminal cysteine phytase fragments retrieved from the Small Tail Han sheep
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Sequence
Percentage of cysteine phytase fragments (%)
identity (%)
cDNA of 0 h
cDNA of 4 h
cDNA of 9 h
cDNA of 16 h
DNA of 9 h
80–100
34.78
0
4.35
5.26
5.88
60–80
0
0
8.70
0
0
50–60
34.78
50.00
43.48
52.63
41.18
40–50
30.43
50.00
43.48
42.11
52.94
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Table 2 Identical cysteine phytase gene fragments from the DNA and cDNA clone libraries*
cDNA of 0 h
cDNA of 4 h
cDNA of 9 h
OTU
OT
OT
cDNA of 16 h
DNA of 9 h
Gen e
Abundan ce
scp1
0-1
scp2
0-2
scp3
0-4
scp5
0-3
U
ce
14.08
4-6
46.48
4-1
(%)
(%)
(%)
Abundanc OTU
ce
79.43
9-3
2.97
16-4 3.39
6.38
9-5
43.56
16-1 27.97
G9-5
ce (%)
e (%)
16-6
G9-9
23.74
9-2 6.34 13.38
4.26 4-4
3
17.82
0.7
1.69
7.91
16-5 5.08 9-1
0-6
0-7
Abundan
U
4-7
16-2 6.34
scp6 scp7
Abundan OTU
b
scp4
Abundan
3 4-3
4.95
2.83
1.41
G9-2 11.01
16-3 5.93 9-4
4.95
42.45 G9-3
1.44
G9-12
1.44
4-1
scp8
9-6 5
0.7
16-8 1.98
G9-7 1.69
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5.04
4-2
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scp9
0.7
scp1
9-2
6.93
16-7 33.9
G9-6
6.47
9-1 G9-10
0
4
scp1
9-1
1.05
1.44
G9-4
1
0
scp1
1.05
4-1
2.16 16-1
0-11
2
0.7
4
0.7
scp1
0
0.85
9-2 4-9
3
G9-29 0.7
scp1
1
1.05
9-2
1.44 16-1
0-17
4
0.7
0
1.05
7
0.85
scp1
0-8
5
9-7 0.7
G9-11 1.05
0.92
scp1
0-13
6
G9-13 0.7
scp1
0.92 4-1
16-1
0-18
7
0.7
scp1
2
0.7
4-1
4
0.85
16-1
0-20
8
0.7
0
0.7
1
0.85
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scp1
4-1 0-25
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9
G9-25 0.7
scp2
1
0.7
0.92
4-1 G9-16
0
6
0.7
0.92
scp2
4-5
1
9-8 0.7
G9-15 1.05
scp2
16-1
0-26
2
0.92
9-1 0.7
1.05
scp2
2
0.85
9-1 1.05
3
G9-26
6
0.92
scp2
16-9
4
Total 14
G9-19 0.85
93.63
14
100
15
91.56
14
95.76
*
The most predominant gene fragments are shown in bold.
†
OTU, operational taxonomic unit.
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0.92 17
100
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FIGURE LEGEND
Fig. 1 Phylogenetic analysis of the partial amino acid sequences of cysteine phytase retrieved from the ruminal genomic DNA and cDNA of Small Tail Han sheep and reference sequences. The microbial sources and GenBank accession numbers (in parentheses) are shown. The tree was constructed using the Neighbor-Joining method in MEGA 4. Branch lengths indicate the relative divergence among the amino acid sequences, and bootstrap values are expressed as percentages from 1,000 replications. Representative genes used for further quantitative analysis are shown in bold
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