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2 Molecular Cloning and Characterization of the Phycocyanin Genes From Spirulina Platensis c1

2 Molecular Cloning and Characterization of the Phycocyanin Genes From Spirulina Platensis c1

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Molecular cloning and characterization of the phycocyanin genes from
Spirulina
platensis C 
I
Miss Wattana Jeamton
B.Sc.
(Agriculture)A Thesis Submitted in Partial Fulfillment of the Requirementsfor the Degree of Master of ScienceBiotechnology ProgramSchool of Bioresources and TechnologyKing Mongkut’s Institute of Technology Thonburi
1997
Thesis Committee
5
 
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Chairman
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Co-Chairman(Asst. Prof.
Suchada
Chaisawadi)
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Member(Assoc. Prof. Dr. Sakarindr Bhumiratana)
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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Member
(Dr. Patcharaporn Deshnium)
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Member
(Asst. Prof. Dr. K. J. Reddy)
ISBN
974-624-075-7
Copyright reserved
 
iiThesis TitleThesis CreditsCandidateSupervisorsDegree of StudyDepartmentAcademic YearMolecular cloning and characterization of the phycocyaningenes from
Spirulina platensis
Cl
12
Miss Wattana JeamtdnAsst. Prof. Dr. Supapon Cheevadhanarak Asst. Prof.
Suchada
ChaisawadiAssoc. Prof. Dr. Morakot TanticharoenMaster of ScienceBiotechnology
1997
AbstractIn this study, three strategies were employed in the isolation of thephycocyanin genes
(cpcBA)
 
from S.
 platensis
Cl.
 
These include cloning phycocyaningenes by inverse polymerase chain technique (IPCR), by screening genomic andpartial library. The phycocyanin genes were successfully obtained from the genomiclibrary. The
cpcA
 
specific probe designed from the conserved amino acid sequencesof 
cpcA
 
genes of various cyanobacteria and one red alga (Cheevadhanarak,unpublished) was used as a homologous probe to screen
hDASHI1
 
genomic DNAlibrary of 
S. platensis
Cl.
One out of twenty positive recombinant
h
clones containingphycocyanin genes was isolated and their sequences were determined. Two openreading frames of 5 16 bp and 486 bp corresponding to
cpcB
 
and
cpcA
 
genes,encoding
p
and a subunits, respectively, were found in the
EcoRV
 
fragment of the
 
.
 
.
 
111
genomic DNA. The
cpcH
 
and
cpcl
 
gene encoding rod-rod linker proteins were alsofound downstream of the
cpcA
 
gene. The identity of the deduced amino acidsequences of 
cpcB
 
and
cpcA
 
genes compared to those of 
Synechocystis
PCC6701,
Synechococcus elongatus,
Pseudoanabaena
 
PCC7409
and
 Agaothamnion neglectum
were 82.5, 8 1.9, 79.6 and 70.9% for
cpcB
 
gene and 79.6, 75.3, 79.6 and 70.9% for
cpcA
 
gene, respectively. For the
cpcH
 
and
cpcI
 
genes, their partial amino acidsequences revealed the highest score of identity with those of 
Synechococcus
PCC6301
as 53.1% and
69.5%,
respectively. Expected chromophore attachment siteswere found at cysteines position 84 of the a-subunit and position 82 and 153 of theP-subunit. DNA sequence and restriction mapping analysis revealed that the obtainedgenes form
cpcBAHI
 
operon with a putative transcriptional start and terminationsites. The putative transcriptional initiation site of this operon was located 290nucleotides upstream from the translation start site of the
cpcB
 
gene. Additionally,the sequences similar to
 Escherichia
coli
 
consensus promoter sequences were alsofound at the putative promoter regions of the
cpcBAHI
 
operon of 
S.
platensis
Cl.
These results suggest that the obtained genes form
cpcBAHI
 
operon andencodes the phycocyanin and rod components of 
S. platensis C 
1.
Keywords:
Spirulina
platensis
 
 /phycocyanin
genelcpcBAHI
 
operon/
cyanobacterium/phycocyanin

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