Cloning Of Antifreeze Protein Gene And Its Integration In Giant Freshwater Prawn (Macrobrachium Rosenbergii)

Antifreeze proteins(AFPs) are special proteins that have the ability to inhibit ice crystal growth, which depresses the freezing point of body fluid, thereby allowing survival at subzero temperatures. AFPs are firstly found in polar fishes, and then isolated from insects and plants. Several attempts have been made to utilize modern transgenic techniques to express AFPs genes in frost-susceptible animals and crops to increase their freezing tolerance, and it has been reported that some transgenic organisms do acquire increased cold-resistance. The giant freshwater pravm(Macrobrachium rosenbergii) is of great economic value and cultured around the world. This prawn has been popular in Chinese mainland due to its delicious taste and abundant nutrition since it was introduced here. Now it has been one of the most important aquatic products in the freshwater cultivation. However, this prawn can't survive at a water temperature lower than 14C, which has seriously limited its cultivation expanding. In order to obtain a new breed of this prawn with increased cold-resistance, we investigated the cloning of a synthetic gene(sbwAFP) based on the primary sequence of the mature spruce budworm(Choristoneura fumiferana) antifreeze protein(sbwAFP) and the integration of sbwAFP into the embryo genomes of giant freshwater prawn by spermatophore-microinjection(SMI), a sperm-mediated gene transfer technique.sbwAFP was assembled by primer overlap extension and amplified using PCR. After that, it was inserted into a plasmid vector pCMV - EGFP - SV40UTR between the cytomegalovirus promoter (CMVp) and an enhanced green fluorescent protein cDNA(EGFP) to yield an expression vector pCMV-SbwAFP-SV40UTR. This recombined plasmid was transferred into the giant freshwater prawn using SMI. Fertilization and hatching of prawns created with SMI were completed as normal. The genomes of SMI-created prawn embryos were analyzed by PCR with a pair of specific primers of sbwAFP. A specific product with a molecular mass of 308bp wasamplified in some of the SMI-created samples, while no amplified band appeared in the samples from prawns injected with PBS and without injection, which indicated that the exogenous sbwAFP was integrated into the SMI-created prawn embryo genomes. Among a gradient of injected plasmid DNA mass, which was from 250ng to 1850ng, the 1850ng SMI-created prawn embryos showed a gene transfer rate of at least 16%.