Please use this identifier to cite or link to this item:
Molecular Cloning of Hemocyanin cDNA and its Expression in Freshwater Giant Prawns, Macrobrachium rosenbergii
|Publication Year :||1998|
在選殖血青素基因方面，以十足目血青素氨基酸保守片段設計出consensus primer pair: Cul123-Cul721，對淡水長臂大蝦肝胰臟進行RT-PCR，得到-598bp大小的cDNA片段，其氨基酸及核酸序列和南美白蝦分別有98％輿75％的相似度。利用此consensus primer pair對其他位於軟甲綱下分屬於口足目，等足目輿十足目的25種類進行RT-PCR，結果均能自其中放大出約600bp預期大小之cDNA片段，以淡水長臂大蝦血青素cDNA製成之非放射性核酸探針進行Southern blot，證實放大之600bp cDNA片段均?血青素基因，故primer pair: Cu1123-Cu1721，確實可作?選殖甲殼類血青素基因之共通核酸引子，並由此推測在軟甲綱血青素基因的第二domain應當相當保守。
在血青素生合成位置的研究方面，以digoxigenin標定之非放射性探針對眼柄，肝胰臟，鰓，肌肉，精巢，卵巢與血球細胞進行Northern blot，結果只有肝胰臟具有血青素mRNA表現，其mRNA大小約2.5kbp，另外以consensus primer pair: Cu1123-Cu2416對上述諸組織進行RT-PCR檢定，亦只有肝胰臟具有血青素mRNA表現，顯示血青素mRNA的表現具有高度的組織專一性。
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 70-80kDa 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 77kDa). 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.
|Appears in Collections:||漁業科學研究所|
Files in This Item:
There are no files associated with this item.
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.