The Construction Transgenic P₀ Grass Carp Of Reovious And Molecular Cloning Of Chordin Gene In Fish And Its Impact On Tail Type In Goldfish

At present, the research of gene function and transgenic is an important direction in the fish breeding field. By studying a master gene, selected new varieties of superior traits. It is advantageous for progress and development to fish farming industry.To explore new methods to prevent the grass carp(Ctenopharyngodo idellus)hemorrhage,the capsid-targeted viral inactivation(CTVI)strategy was developed in present study. By combination with Cyprinus carpio heat shock protein 70 or Xenopus EF1α promoters, two different transgenic plasmids p Tgf2-EF1α-VP3-SN and p Tgf2-Hsp70-VP3-SN were constructed harboring with efficient Tgf2 transposon element. Both transgenic plasmids contain open reading frame(ORF) fusion withGCRV caspsid protein VP3 and Staphylococcus nuclease(SN).The transgenic grass crap was produced by co-injection p Tgf2-EF1α-VP3-SN or p Tgf2-Hsp70-VP3-SN plasmids into 1-2 cell fertilized eggs with in vitro synthesized Tgf2 transposase m RNA. The PCR and sequencing analysis have proved that anti-GCRV transgenic systems have successfully been integrated into the genome of grass carp. The transgene positive rates of p Tgf2-EF1α-VP3-SN or p Tgf2-Hsp70-VP3-SN plasmids are 40.2% and 37.0%, respectively. Our results demonstrated Tgf2 transposon can efficiently mediated transgenesis in grass carp for fusion ORF of GCRV caspid protein VP3 and SN. Total 120 transgenic grass crap individuals have been obtained in the present study. The construction of anti-GCRV transgenic P0 will provide the materials for future transgenic breeding to prevent grass carp hemorrhage.The result of isolation of Chordin in Megalobrama amblycephala is as follows: The full length of Chordin is 3736 bp, which 2829 bp ORF which encodes 942 amino acids. Chordin includes 18 amino acid signal peptide and 924 amino acid mature peptide. Like Homo sapiens and zebrafish,the mature polypeptide of in Chordin in Megalobrama amblycephala also contains tworegions:CHRD domain and Vwc domain. When compared to human, goldfish A/B and zebrafish similarity in coding region, blunt snout bream Chordin has more than 68%, 89%, 88% and 68% respectively. So clearly, the blunt snout bream Chordin has a highly conservation in evolution. Chordin m RNA was detected by RT-PCR throughout embrygenesis and showed different expression patterns. The expression level of Chordin stabilized from 0hpf to 32 hpf. The expression level of Chordin gradually decreased in the 36 hpf. In adult fish, Chordin m RNA was transcribed in multiple tissues, especially in the brain which had a highest expression. By in situ hybridization, Chordin m RNA was observed mainly in the tail, especially in the 12 pdf embrys. Brain Chordin transcript level was increased significantly after 2 to 6 days of starvation, especially in the 4 days was decreased, and they were restored rapidly after refeeding. The levels of brain Chordin m RNA decreased with the increase of growth hormone injection. By molecular cloning and functional characterization of Chordin in Megalobrama amblycephala, which could been used in the megalobrama amblycephala thoroughbred breeding.In addition, this study also researched the correlation between the genotype of Chordin and fish tail type(single-tail/twin-tail). There are few reports on the determining factors of the caudal fin phenotypic traits. Known as one of the important famous aquarium fish in China, the tail characters of goldfish is a direction domestication. Goldfish growth cycle is short, but the tail fin traits has a bigger variation. There was a few reports about the determining factor of tail fin in aquatic animal. In which the chordin A gene plays an important role in the formation of goldfish caudal phenotype. In the study, molecular biology techniques were used to clarify the chordin A gene determined mechanism of tail type. Our result demonstrated that the phenotype was twin-tail when the sixth base A muted to G and the sixteenth base G muted to T in the 18 bp sequence of chordin A gene. Furthermore, the genotype of twin-tail goldfish was double tail homozygous site. Blasting this specific base nucleotide sequence analysis in many fish, this sequence has a highly conservation. By in situ hybridization in single-tail and twin-tail embryos, the expression level of Chordin A m RNA in single-tail embryo was higher than twin-tail. Myo D m RNA was gradually reduced from the back to the tail in single-tail embryo. However, Myo G m RNA was gradually increased from the back to the tail in twin-tail embryo. Ntl m RNA was observed justly in the twin-tail embryo. The expression levels of Bmp4 m RNA in both single-tail and twin-tail embryos were high. Through the CRISPR- Cas9 system of Chordin A knockout, the experimental results show that the a allele Chordin A gene mutation, controlled by another allele. In the process of the formation of single or twin-tail, egg quality especially the concentration of Chordin A m RNA plays an important role, it can control the formation of twin-tail. The research shows that Chordin A gene mutation is the necessary condition of twin-tail but not sufficient condition.