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Preliminary Study of EDA、EDAR Gene Cloning and Localization of Expression in Cyprinus Carpio

Author: WangYangYang
Tutor: SunXiaoWen
School:
Course: Biochemistry and Molecular Biology
Keywords: carp scales RACE in-situ hybridization
CLC: S965.116
Type: Master's thesis
Year: 2014
Downloads: 3
Quote: 0
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Abstract


As the most important Cyprinidae, carp is one of the most widely freshwatercultivated species in the world. Mirror carp and common carp are two breed in carp.In fish farming, the scales are characterized by relatively easy to find. Currently, verylittle research has been on the scales, especially in terms of carp. Long-term studyfound that mirror was significantly better than other aspects of common carp in meat,growth rate and so on. And the most obvious trait of the both is the skin covered withscales. More than that, it can not be ignored in disease resistance and studied haveshown that fish scales have common evolutionary ancestor with mammalian skinappendages such as hair, sweat glands or teeth. Therefore, we started with carp scales.Screen relevant factors by differential screening of developmental in scales. Thisstudy selected two genes and improved the study of fish especially in mammal hair,sweat glands, etc by cloning, sequencing and functional analysis.The Ectodysplasin-A (EDA) was firstly found in mammal, it plays vital roles indevelopmental events of the skin appendages. The screening of transcripts of skinfrom common carp and the Germany mirror carp by Genefishing differential displayfound EDA is one of the targeted different expressed in both materials, the partialfragment of EDA gene was recovered from differential expressed products bysequencing. The specific primers contained the full length of EDA were designed withthe reference sequence of EDA EST in our Carp database. The mirror carp EDA CDSconsisted of1062base pair nucleotides which encodes a354amino acid protein, ithas a receptor-binding region. The alignment result shows that the carp shares thehighest similarity (92.76%) with the zebrafish. However, it has the relative lowersimilarity with chicken (46.61%), human (50.51%), mouse (50.76%) due toevolutionarily distant relationships. The5’-UTR regulation region of EDA shares thehigh similarity with the zebrafish (92.98%), but varies significantly with human (only23.36%), mouse (31.07%).Using same methods, we achieved the part of EDAR fragments in mirror carp. We found that EDAR CDS consisted of1389base pair nucleotides which encodes a462amino acid protein, it has a signal peptides, a Tumor necrosis factor receptorregion, a transmembrane region and death domain. The alignment result shows thatthe carp shares the highest similarity (88.31%) with the zebrafish. However, it has therelative lower similarity with Xenopus laevis (64.88%) chicken (63.79%), human(64.36%), mouse (63.50%) due to evolutionarily distant relationships. The5’-UTRshares the high variation, even with the medaka (24.73%) and zebrafish (23.78%).Also in the3’-UTR varies significantly with medaka (35.49%). Let alone withXenopus laevis (11.73%and9.42%), human (6.50%and8.05%), mouse (10.04%and9.37%).The in-situ hybridization results show that the two genes unique expressionsignal was detected in skin matrix which the scale formed, however weak or noexpression signal was detected in other positions on skin, later disappeared in fulldeveloped scale body, all of these results show that this gene may involve theinitiation of the scales not the maintenance of the scale pattern formation.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquaculture technology > A variety of fish farming > Freshwater fish > Carp
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