Molecular Cloning Of CTFG Genes And The Study On The Construction And Transgenic Efficiency Of PB Transposon And PB-Tgf2Transposition System In Megalobrama Amblycephala

Blunt snout bream （Megalobrama amblycephala） has an importanteconomic statusin our freshwater fish,but it is an extremelyhypoxia-ntolerant fish. It will cause mass death and great economic lossesunder hypoxicconditions, which restricts the improvement of its production.Explore the effect the function of the gene for aquatic animals is theimportant content of the genetic breeding.Transposons play a crucial role interms of obtaining and study t offunctional genes,with potential applicationprospects in molecular breeding of aquatic animals.Connective tissue growth factor （CTGF） which is a cysteine-richsecretory peptide，also known as CCN2,is a member of the CCN familyofmodular matricellular proteins.CTGF contains an N-terminal secretory peptide, followed by four multi-functionaldomains with a diverse array ofbinding partners that potentially impact multiplesignalingmechanisms.Research shows that interactions between CCN2andsome binding partners mediate the effects of CCN2on cellproliferation,survival, differentiation, adhesion, migration, and ECMproduction.Because CTGF contains IGFBP domain, and the intron ofIGFBP-1contains hypoxia-inducible factor HRE, therefore we speculatethat CTGF may also be involved in associated molecular mechanisms ofhypoxia response inblunt snout bream.We report herein on the isolation and characterization of duplicatedCTGF genes （Ctgfa and Ctgfb）inMegalobrama amblycephala.Blunt snoutbream Ctgfa and Ctgfb cDNAs are2153bp and1467bp in length,respectively. Their ORFs are1032bp and1056bp, which encode343and351amino acids, respectively. Like their human and zebrafish orthologs,blunt snout bream Ctgfa and Ctgfb mature peptides also contain fourregions: IGFBP, VWC, TSP₁and Cys_knot, in which the numbers oftheircysteine groups are11,10,5and11, respectively. When compared tohuman and zebrafish, blunt snout bream Ctgfa and Ctgfb have more than55%similarity in the coding region. Both Ctgfa and Ctgfb mRNAs weredetected by RT-PCR throughout the embryogenesis and showed differentialexpression patterns. The expression level of Ctgfa gradually decreased in the0-12hpf stage and significantly increased from16hpf, then itstabilizedfrom32hpf.The expression level of Ctgfbwasweak,constantandthe difference was notsignificant from0-20hpf,then itgradually increased from24hpf. In adult fish, Ctgfa mRNAs weretranscribed in multiple tissues except in heart, liver and gonads, whileCtgfb mRNAs were detected in all tissues. By in situ hybridization,CtgfamRNAs was observed in in the brainand spinal cord, whereas CtgfbmRNAs were expressed mainly in the spinal cord.The amount of theexpression in Ctgfb is less than that in Ctgfa.Meanwhile,we use the PiggyBac transposon and PB-Tgf2transpositionsystemto carry out transgenic efficiency in Megalobramaamblycephala.The two donor vector plasmidspPBs-EF1α-eGFP,pPBs-Tgf2-RPrmP-eGFP and the helper plasmidpCS2-PBTP were constructed.And the donor vector withPB transposasemRNA were microinjection to fertilized egg together.In adult fish, PCRresults demonstrated that integration efficiency of PiggyBac transpositionsystem and PB-Tgf2transposition system were53.04%and32.14%respectively in M. amblycephala genome. Our data suggest thatPiggyBactransposon and PB-Tgf2transposition can be efficiently mediatedgene insertion in M. amblycephala, which could been used in insertionalmutagenesis in M. amblycephala.