Construction And Transference Of Carassius Auratus Mx Gene Recombinant Into Embryos Of Grass Carps

The coding sequence of Mxl gene of Carassius auratus was cloned under the control of carp β-actin gene promoter by restructuring and molecular cloning technology. The recombinant was then injected into the fertilized eggs of grass carps by the method of micro-injection.It obtained mass of all-fish transgenic grass carps. This experiment provided research materials and ideas for disease-resisted breeding for grass carps.In order to amplify the open reading frame(ORF) of Carassius auratus Mxl gene, a pair of Primers were Designed according to the GenBank sequence of Carassius auratus Mx1gene (AY303813.1).The cloned seqence was connected with pEASY-T5Zero cloning Vector and transformed into Trans1-T1.Then chose colony to submerged cultrue, the bacteria liquid was tested by colony PCR and send to sequencing. Then the plasmid was extraction from the bacteria and treated with BamHI and EcoRl, subcloned into pUC118-cap which was also treated with BamHI and EcoRl.The recombinant plasmid was transformed into Escherichia coli DH5a, then had the blue-white selection and chose the white colony to submerged culture.Then extract the plasmid from the bacteria and treated with BamHI and EcoRl. It verified the correctness of the recombinant, then an all-fish Mx1gene recombinant was constructed, named JMxl-cap.The recombinant gene was transferred into fertilized zygotes of grass carps by micro-injection after Pvu Ⅱ linearing.A group of all-fish transgenic grass carps were obtained.Polymerase chain reaction(PCR) technology and Southern blot were carried out to verified transgenic grass carps.It turned out that about39%of treated grass carps fry (two months) contained exogenous gene detected by PCR analysis. In Southern blot analysis, transformations got positive signals. The results showed that all-fish Mx1gene had integrated into the genome of grass carps.