碩士 / 國立臺灣大學 / 漁業科學研究所 / 86 / Hemocyanin is a copper-containing respiratory pigment occurred
in two phyla of invertebrates:
the Arthropoda and the Mollusca. In Crustacean, six functional
subunits(-mer) around 70-80 kDa were
aggregated to form the basic quaternary structure, 12-mer or
even 24-mer could also be found. In
freshwater giant prawns, Macrobrachium rosenbergii, hemocyanin
was composed of two subunits (85
and 77 kDa). In order to verify whether the two hemocyanin
subunits were coded by different gene
or just the result of post-translational modification, total
RNA from hepatopancreas was translated
in vitro with rabbit reticulocyte lysate and immunoprecipitated
by hemocyanin antiserum. N-terminal
amino acid residues of each subunit was also sequenced. The
results indicate that the two hemocyanin
subunits were coded by different gene in Macrobrachium rosenbergii.
Although Crustacean hemocyanin has been extensively studied on
the protein level, hemocyanin
genes were elucidated only in Penaeus vannamei and Cancer
magister (Order Decapoda). Whether hemocyanin
genes are conserved in Decapoda or even in Malacostraca is
waiting to be verified. PCR-based strategy
with one set of consensus primer pair: Cu1123-Cu1721 designed
according to the copper-binding domain
was adopted. The predicted 600 bp cDNA fragments amplified from
25 different species which represented
three different orders (Stomatopoda: 1 species, Isopoda: 1
species and Decapoda: 23 species) were further
confirmed by Southern blot with non-radioactive DNA probe
labeled with digoxigenin. Thus this PCR methodology
using the consensus primer pair is widely applicable for
amplifying Crustacean hemocyanin genes and the
copper-binding domain of hemocyanin genes is highly conserved in Malacostraca.
The hemocyanin biosynthesis site(s) were reported in
hepatopancreas in many decapoda species but
whether the extra-hepatic tissue could also producing
hemocyanin is waiting to be verified in Decapoda.
In this study, Northern blot and RT-PCR strategies were taken
to investigate the possible hemocyanin producing
site at the molecular level. The results show that hemocyanin
mRNA was only expressed in hepatopancreas
but not in eyestalk, gill, muscle, testis, ovary and hemocytes.
Ontogenic expression of hemocyanin was studied by PCR-based
strategy with one set of consensus
primer pair: Cu1123-Cu2416. The results indicate that
hemocyanin mRNA could not be detected in ovary at
any gonadal stage and hemocyanin mRNA was expressed
endogenously by the embryos from the fourth days after
fertilization to hatching out.
| Identifer | oai:union.ndltd.org:TW/086NTU00451002 |
| Date | January 1998 |
| Creators | Hsiao, Chung-Der, 蕭崇德 |
| Contributors | Lou, Show Wan, Tsai, Huai-Jen, 羅秀婉, 蔡懷楨, --- |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 82 |
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