The Construction And Detection Of Transgenic Spinibarbus Sinensis With Growth Hormone Gene

The expression recombinant plamid pFV2cfGHc was constructed from growth hormone gene which was cloned from the channel catfish cDNA library, theβ-action promoter of carp and the polyadenylation signal from the salmon growth hormone gene, and other controlling elements. The recombinant plasmid pFV2cfGHc was transmitted into the fertilized eggs of the Spinibarbus Sinensis through Sperm-mediated gene transfer. Total DNA of 423 transgenic Spinibarbus Sinensis was abstracted, and growth hormone gene were detected by PCR. The results showed that the growth hormone gene of the channelfish had been transmitted into the genome of the Spinibarbus Sinensis and the integration rate is 17. 26%. At the same time, the expression of, growth homone gene in positive Spinibarbus Sinensis was detected by RT-PCR.In the experiments we compare four different fish genomic DNA extraction methods included genomic blood DNA purification kit, genomic tissue DNA purification kit, the manual isolation of genomic blood DNA and the manual isolation of genomic tissue DNA. At last, we concluded that the manual isolation of genomic blood DNA is the best method to get the fish genomic DNA, and we use this method to get the total DNA of 423 transgenic Spinibarbus Sinensis. Then, the experimental parameters was optimized, and the optimal experiment conditions were ascertained(1.5mmol/L Mg²⁺, 25ng template, anneal at 69℃). Under the optimal experiment conditions, total DNA of 423 transgenic Spinibarbus Sinensis was abstracted, and growth hormone gene were detected by PCR.The results showed that the growth hormone gene of the channelfish had been transmitted into the genome of the Spinibarbus Sinensis and the integration rate is 17.26%.In the experiments, we used guanidinium isothiocyanate and TRI REAGENT purification kit two RNA isolation methods to extracting total RNA from the Spinibarbus Sinensis brain tissue . RNA extracted by this method is not polluted by DNA and can meet the demands of research in gene expression. Using RT-PCR with the two special primers, the expression of growth homone gene in positive Spinibarbus Sinensis was detected.