Download PHYCOCYANIN ALPHA AND BETA SUBUNITS OF Anabaena

1
PHYCOCYANIN ALPHA AND BETA SUBUNITS OF Anabaena
2
siamensis TISTR8012
3
4
Wanwisa Suphap and Mariena Ketudat-Cairns*
5
6
Abstract
7
Phycocyanin (PC) is a non-toxic biliprotein consisted of  and ß subunits that act as
8
photoharvesting pigment in various cyanobacteria. The PC subunits of Anabaena
9
siamensis TISTR8012 that was isolated from Thai rice paddies have not been studied.
10
The genomic DNA of A. siamensis TISTR8012 was used as template for cpcA; alpha
11
subunit (α subunit) and cpcB; beta subunit (β subunit) amplification. The primers for α
12
and β subunit amplification were designed from the alignment of α and β subunit of A.
13
variabilis ATCC 29413, Arthrospira platensis, Spirulina maxima, A. kisseleviana, A.
14
lemmermannii, A. flos-aquae and A. planktonica. In this paper the PC  and ß subunits
15
from A. siamensis TISTR8012 were isolated, sequenced and submitted to NCBI
16
database accession number EU815327 and EU815328, respectively.
17
18
Keywords: Anabaena siamensis TISTR8012, antioxidant and phycocyanin
19
20
School of Biotechnology, Institute of Agricultural Technology, Suranaree University of
21
Technology, 111 University Avenue, Maung District, Nakhon Ratchasima 30000, Thailand,
22
Tel. 0-4422-4355
23
Fax. 0-4422-4154 E-mail: [email protected]
24
*Corresponding author
25
1
1
Introduction
2
Anabaena siamensis is a member of cyanobacteria isolated from rice paddy field in
3
Thailand. Recently this strain has been used in the Thai agricultural sectors as an algal bio-
4
fertilizer for rice fields. It shows high capacity to fix atmospheric N2 and subsequently
5
increase the rice growth rate (Phunpruch et al., 2006). Phycocyanin (PC) is a non-toxic
6
biliprotein consisted of  and ß subunits that act as photoharvesting pigment in various
7
cyanobacteria.
8
PC from many cyanobacteria has been reported to work as antioxidant scavenger. For
9
example the recombinant PC β subunit from Anabaena PCC7120 can inhibit cell
10
proliferation and induced apoptosis by promoting cytoskeleton depolymerization and
11
activating the caspase activities that are associated with the extrinsic cell death pathway
12
(Wang et al., 2006). The Cyanobacterial C-phycocyanin (C-PC) holo subunit (holo-CpcA)
13
has been successfully expressed as a fluorescent holo-CpcA in E.coli BL21. The heme
14
oxygenase 1 and 3Z-phycocyanobilin: ferredoxinoxidoreductase enzymes required for
15
conversion of heme to phycocyanobilin (PCB), and cpcA encoding alpha subunit along with
16
cpcE and cpcF both of which necessary and sufficient for the correct addition of PCB to
17
CpcA to occur blue colour were also cloned in the same plasmid (Xiangyu et al., 2007).
18
Native PC has variety of properties, however it is difficult to purify. In this paper the  and ß
19
subunits from A. siamensis TISTR8012 were constructed to produce recombinant PC for
20
further used in colorance and antioxidant test, which should be easier to purified than the
21
native PC.
22
However, no study on sequence of PC  and ß subunits of A. siamensis TISTR8012
23
has been reported. In this paper, two pairs of primers for cpcA and cpcB genes for α and β
24
subunits were designed from A. variabilis ATCC29413, Arthrospira platensis, Spirulina
25
maxima, A. kisseleviana, A. lemmermannii, A. flos-aquae and A. planktonica. The primers
2
1
were used to amplify the cpcA and cpcB genes from A. siamensis TISTR8012 and cloned into
2
cloning vectors, then sequenced and submitted the sequence to NCBI database and the
3
accession
number
EU815327
and
EU815328
were
received,
respectively.
4
5
Materials and Methods
6
Cyanobacterial strain
7
A. siamensis TISTR8012 requested from the Thailand Institute of Scientific and
8
Technological Research (TISTR) was cultured in BG11 medium.
9
Primer design
10
Gene specific primers were designed from the alignment of α and β subunits of 7
11
cyanobacteria (A. variabilis ATCC29413, Arthrospira platensis, Spirulina maxima, A.
12
kisseleviana, A. lemmermannii, A. flos-aquae and A. planktonica) for PCR amplification of
13
cpcA and cpcB genes of A. siamensis TISTR8012. Anabaena billiprotein phycocyanin
14
operon accession number X 05239 was used as reference sequence (Belknap and Haselkorn,
15
1987).
16
Genomic DNA extraction
17
A. siamensis TISTR8012 grown in BG 11 medium for 14 days were collected by
18
centrifugation at 14,000 x g, room temperature for 10 min. Genomic DNA were extract by
19
phenol: chloroform: isoamylalcohol method as previously described (Golden et al., 1988).
20
Amplification of cpcA and cpcB genes of PC
21
The Polymerase Chain Reaction (PCR) was used to amplify the cpcA and cpcB genes
22
of the α and β subunits of phycocyanin. The cpcA gene primers were used to amplify the α
23
subunit and the cpcB gene primers were used to amplify the β subunit from A. siamensis
24
TISTR8012, respectively.
25
3
1
Cloning of α and β subunits of PC
2
The PCR products were cloned into pENTR/D-TOPO (invitrogen: FIG.1) cloning
3
vector and transformed into E. coli (strain DH5α) by electroporation technique (1,800 v) and
4
plated on LB agar contain 50 g/ml of kanamycin for selection. The plasmid was extracted
5
using QIAprep Spin Miniprep Kit (Qiagen) from the transformant clones and further used for
6
DNA sequencing analysis.
7
8
Results and Discussion
9
The extracted genomic DNA (gDNA) of A. siamensis TISTR8012 by phenol:
10
chloroform: isoamylalcohol were shown to be of high quality with high molecular weight on
11
1% agarose gel in FIG.2.
12
The gDNA was used as template for PCR reaction with primers shown in Table 1.
13
The PCR product of 480 bp and 500 bp were seen for the cpcA and cpcB genes, respectively
14
(FIG.3). The PCR products were cloned into pENTR/D-TOPO and transformed into E. coli
15
DH5α. Recombinant plasmids were prepared from several transformant clones and sequence.
16
The sequencing results indicated that the clones were α subunits with 99% identity to
17
Anabaena variabilis ATCC29431 (CP000117), Anabaena PCC7120 (AF178757) and
18
Anabaena 7120 biliprotein (X05239). The results of β subunits with 100% identity to
19
Anabaena variabilis ATCC29431 (CP000117), Anabaena 7120 biliprotein (X05239) and
20
Anabaena PCC7120 (AF178757) (FIG.4). The sequence of both α and β subunits of A.
21
siamensis TISTR8012 were submitted to NCBI database. The accession number EU815327
22
and EU815328 were obtained for α and β subunits, respectively.
23
FIG.5 indicate the nucleotide sequence of cpcA gene and the deduce amino acid
24
sequence that contained the conserved cysteine at position 85 (α85) whereas in Spirulina
25
platensis (Padyana et al., 2001), Cyanidium caldarium (Stec et al., 1999) and Polysiphonia
4
1
urceolata (Jiang et al., 2001) contained cysteine at position 84 (α84). FIG.6 indicate the
2
nucleotide sequence of cpcB gene and the deduce amino acid sequence that contained the
3
conserved cysteine at position 83 and 154 (β83 and β154) whereas Spirulina platensis
4
(Padyana et al., 2001), Cyanidium caldarium (Stec et al., 1999) and Polysiphonia urceolata
5
(Jiang et al., 2001) contain the conserved cysteine at position 84 and 155 (β84 and β155)
6
Phylogram of the phycocyanin β subunit indicated that the PC β subunit of A.
7
siamensis TISTR8012 is closely related to the PC β subunit of A. variabilis ATCC19413
8
(FIG.7) However the α subunit of A. siamensis TISTR8012 sequence is more similar to
9
Arthospira platensis than A. variabilis ATCC19413 (FIG.8)
10
In conclusion the cpcA and cpcB genes from A. siamensis TISTR8012 has been cloned,
11
sequenced and the sequence have been submitted to GenBank with the accession number
12
EU815327 and EU815328, respectively.
13
14
Acknowledgement
15
WS was supported by SUT Outstanding graduate student scholarship.
16
17
References
18
[1]
19
Nucleotide sequencing and transcriptional analysis of uptake hydrogenase genes in the
20
filamentous N2-fixing cyanobacterium Anabaena siamensis. Journal of Applied
21
Phycology 18: 713–722.
22
[2]
23
subunit of C-phycocyanin inhibits cell proliferation and induces apoptosis.
24
Letters. 1-9.
Phunpruch, S., Baebprasert, W., Thongpeng, C., and Incharoensakdi. A. (2006).
Wang, H., Liu, Y., Gao, X., Carter, C.L. and Liu, Z.R. (2006). The recombinant β
Cancer
5
1
[3]
Xiangyu, Guan., Song, Qin., Zhongliang, Su., Fangqing, Zhao., Baosheng, Ge.,
2
Fuchao, Li., Xuexi, Tang. (2007). Combinational Biosynthesis of a Fluorescent
3
Cyanobacterial Holo-α-Phycocyanin in Escherichia coli by Using One Expression
4
Vector. Applied Biochemistry Biotechnology. 52-59.
5
[4]
6
expression of the phycocyanin operon of the cyanobacterium Anabaena. The EMBO
7
Journal. 6 (4): 871-884.
8
[5]
9
(1988). Deletion of a 55-Kilobase-Pair DNA Element from the Chromosome during
10
Heterocyst Differentiation of Anabaena sp. Strain PCC 7120. Journal of Bacteriology.
11
170 (11): 5034 – 50
12
[6]
13
(2001). Crystal Structure of a Light-Harvesting Protein C-Phycocyanin from Spirulina
14
platensis. 282: 893-898.
15
[7]
16
from Cyanidium caldarium Provides a NewP erspective on Phycobilisome Assembly.
17
76: 2912-2921.
18
[8]
19
of R-Phycocyanin and Possible Energy Transfer Pathways in the Phycobilisome. 81:
20
1171-1179.
Belknap, W.R. and Haselkorn, R. (1987).
Cloning and light regulation of
Golden, J.W., Carrasco, C.D., Mulligan, M.E., Schneider, G.J. and Haselkorn, R.
Padyana, A.K., Bhat, V.B., Madyastha, K.M., Rajashankar, K.R. and Ramakumar, S.
Stec B., Troxler R.F. and Teeter M.M. (1999). Crystal Structure of C-Phycocyanin
Jiang T., Zhang Ji-ping., Chang Wen-rui. And Liang., Dong-cai. (2001). Crystal Structure
21
22
23
24
25
26
6
1
FIG.1 Map and Features of pENTR/D-TOPO cloning vector
2
3
FIG.2 Agarose gel electrophoresis of genomic DNA (gDNA) extracted by phenol: chloroform:
4
isoamylalcohol method with the size of more than 10 kb (lane 1-3; triplicate)
5
6
FIG.3 Agarose gel electrophoresis of amplified PCR product of cpcA and cpcB genes. MK:
7
100 bp marker, lane 1 - lane 3: PCR product 500 bp of cpcB gene and lane 4 - 5 PCR
8
product 480 bp of cpcA gene.
9
10
FIG.4 The DNA sequencing of cpcA and cpcB genes in pENTR/D-TOPO when compared
11
with other α and β subunits of other cyanobacteria (NCBI blast).
12
13
FIG.5 The nucleotide sequences and deduces amino acid sequence of PC α subunit.
14
Nucleotide and amino acid residues are arranged from 5’end and the initiator methionine
15
respectively. The underline sequences indicate the forward and reverse primers. The solid
16
triangle indicates the cys 85 that conserved amino acid.
17
18
FIG.6 The nucleotide sequences and deduces amino acid sequence of PC β subunit.
19
Nucleotide and amino acid residues are arranged from 5’end and the initiator methionine
20
respectively. The underline sequences indicate the forward and reverse primers. The solid
21
triangles indicate the cys 83 and 154 that are conserved amino acids.
22
23
FIG.7 Phylogram of PC beta subunit, the accession number from AAT97341; A. flos-aquae,
24
AAT97309; A. kisseleviana, AAT97337; A. lemmermannii, AY702212; A. planktonica,
25
X05239; Anabaena sp. PCC7120, AAL66231; Spirulina maxima, DQ406671; Arthrospira
26
platensis, EU815328; A. siamensis TISTR8012 and YP323436; A. variabilis ATCC 29413.
7
1
FIG.8 Phylogram of PC alpha subunit, the accession number from AAL66232; Spirulina
2
maxima, DQ406671; Arthospira platensis, X05239; Anabaena sp. PCC7120, EU815327; A.
3
siamensis TISTR8012, AAX37370; A. lemmermannii, AAX49574; A. variabilis ATCC
4
29413, AAT97279; A. planktonica, AAT97309; A. kisseleviana and AAT97341; A. flos-aqua
5
6
Table 1 the oligonucleotides primers for DNA amplification base on the sequence from
7
Anabaena 7120 biliprotein (X05239).
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
8
9