Research On The Technology Of Transgene And Gene Knockout Of Pelteobagrus Fulvidraco

Yellow catfish(Pelteobagrus fulvidraco Richardson)is one of the most important freshwater farmed species in China.However,its small size and slow growth rate limits its commercial value.Because genetic engineering has been a powerful tool to develop and improve fish traits for aquaculture,we performed transgenic and gene knock out research on yellow catfish in order to increase its size and growth rate.First,we compared the transgenic construct deliver method of sperm mediated transfer with that of microinjection.Then,we transferred the trangenic reporter constructor of fusing the 5’-flanking sequence upstream of the initiation codon of yellow catfish β-actin gene to coding sequence of eYFP and obtained one transgenic yellow catfish(Tg(beta-atin:eYFP).Third,by microinjecting "all-fish"growth hormone gene transgenic construct,we obtained 160 "all-fish" growth hormone gene transgenic founders and one "all-fish" growth hormone transgenic yellow catfish.At the end,to create sterile yellow catfish,CRISPR/Cas9 system were performed and gnrh gene knockout yellow catfishes carrying one gnrh1 mutated allele(gnrh1+/n-)one gnrh2 mutated allele(gnrh2+/-)or one gnrh1 mutated allele plus one gnrh2 mutated allele(gnrh1+/-;gnrh2+/-)were obtained.These results showed that we have established a yellow catfish genome modification platform that lays the foundation for further molecular breeding to create new strains of yellow catfish with large body size and rapid growth.1.To choose a transgenic method with higher efficiency to integrate the transgenic component into the yellow catfish genome,we compared the transgenic construct deliver method of sperm mediated transfer with that of microinjection.By microinjecting the transgene construct Tg(beta-actin:eYFP)of the proximal promoter fused to yellow fluorescent protein(eYFP)reporter gene into zebrafish embryos,we found that the promoter could drive the reporter to express in embryos at early development.However,no yellow fluorescent signal was found in yellow catfish embryos when the transgenic construct was delivered to yellow catfish embryos by sperm mediated transfer.Encouragingly,the yellow fluorescent signal was found in embryos when the Tg(beta-actin:eYFP)was microinjected into yellow catfish embryos.Totally,451 microinjected yellow catfish embryos were found to express yellow fluorescent signal including 77 broadly expressed.Taken together,the results showed that the microinjection is more effective than the sperm mediated transfer method.2.To make a transgenic yellow catfish,we constructed two trangenic reporters by fusing the 1017 bp 5 ’-flanking sequence upstream of the initiation codon of yellow catfish β-actin gene to coding sequences of mCherry and eYFP,respectively.The two constructs(ycbeta-actin:mCherry)and Tg(beta-actin:eYFP)were then microinjected into zebrafish embryos or yellow catfish embryos at 1-cell stage to produce transgenic founder animals.Screening the offspring of five transgenic zebrafish founders of Tg(ycbeta-actin:mCherry),three lines of transgenic zebrafish were obtained respectively.Analyzing the expression patterns of the reporter genes in transgenic zebrafish(Tg(ycbeta-actin:mCherry)),we found that the reporters were broadly expressed in embryos and adults.Screening the progeny of 19 yellow catfish founders of Tg(beta-actin:e YFP),one transgenic yellow catfish(Tg(beta-actin:eYFP))were obtained.Analyzing the expression patterns of the reporter genes in transgenic yellow catfish(Tg(beta-actin:eYFP)),we found that the reporters were broadly expressed in the juvenile of yellow catfish.In summary,we have established a platform to make transgenic yellow catfish using the proximal promoter of its own β-actin gene.The results will help to create transgenic yellow catfish using "all yellow catfish" transgene constructs.3.By micro injecting "all-fish" growth hormone gene transgenic construct with 2105 bp long comprising of 1017 bp of yellow catfish β-actin proximal promoter,603 bp complete coding sequence and 485 bp 3’-UTR of yellow catfish growth hormone gene,we obtained 160 "all-fish" growth hormone gene transgenic founders of yellow catfish in total.Screening 1478 F1 offspring derived from 12 founders created in the second batch,we successfully obtained one "all-fish" growth hormone transgenic yellow catfish.These results showed that we have established a yellow catfish transgenic platform that lays the foundation for further molecular breeding to create new strains of yellow catfish.4.To guarantee the bio-safety of transgenic yellow catfish,we performed genome editing research to create sterile yellow catfish.Performing CRISPR/Cas9 on yellow catfish,we obtained gene knockout yellow catfishes carrying one gnrhl mutated allele(gnrh1+/-),one gnrh2 mutated allele(gnrh2+/-)or one gnrh1 mutated allele plus one gnrh2 mutated allele(gnrh1+/-;gnrh2+/-).By microinjecting the RNA mixture containing active sgRNA-gnrhl,active sgRNA-gnrh2 and Cas9 mRNA into fertilized eggs of yellow catfish,we established the founder groups of yellow catfish presumptively carrying mutated alleles of gnrhl and/or gnrh2.Screening the F1 offspring derived from the 5 male FO yellow catfish randomly selected from the founder groups,we obtained 51 mutated yellow catfish including 21 gnrh1+/-,20 gnrh2 and 10 gnrh1+/-:gnrh2+/-yellow catfish.The results lay the foundation for creating sterile yellow catfish and therefore prevent the genome modified yellow catfish from polluting the wild population gene bank.