Gene Cloning And Function Assay Of Antifreeze Protein From Several Desert Beetles In Xinjiang

Antifreeze proteins (AFPs),also named thermal hysteresis proteins(THPs)can cause a non-colligative depression of the freezing point of water without changing the melting point, thus produce a temperature difference referred to thermal hysteresis. AFPs are thought to function by inhibiting the growth of small ice crystals, changing their structures and inhibiting the re-crystalization of ice crystal by adsorbing to the surface of ice with hydrogen bonds. AFPs have been isolated from many other organisms such as insects, plants, bacteria and fungi since it was first discovered from polar marine fish in1969. Fish AFPs were studied though extensively, scientists found that the highest thermal hysteresis activity (THA) tested for all AFPs is from insects,which is 10～100 times higher than the fish AFPs and much higher than plant AFPs . Therefore the insect antifreeze protein might be fully applied to many fields such as the storage of oocytes, spermatozoa and embryos, especially in organ cryopreservation for transplantation. It is prosperous to improve plant cold resistance by transferring AFP genes into plants and thus expand their living environment, and raise their productivity. The main task of this study is to clone antifreeze protein genes from three kinds of desert insects, to express antifreeze protein both in prokaryotic and eukaryotic cells, and then to detect the biological activity of these expressed antifreeze proteins as well as to construct a recombinant vector for transgenic plants for the further sdudy. Firstly, PCR primers for amplifing AFP core fragment were designed according to AFPs conservative region published in GenBank between different insects .Total RNA was isolated from the insect treated at 4oC for 24 hours, and cDNAs were synthesized by reverse transcription. The generated core fragments of AFPs were cloned into pMD18-T cloning vector, and positive clones were then checked with restriction enzyme digestion and further identified by sequence analysis and alignment in GenBank.. The results showed that the cloned core fragments were the correct insect AFP genes. With a set of primers designed based on the sequence of the cloned core fragments, the 3'and 5'ends of the AFP gene were obtained by 5'and 3'rapid amplification of cDNA ends (RACE), respectively. Combined with the sequences of the 3'ends, the 5'ends and the core cDNA, the full-length sequence of the cDNA was assembled theoretically.To obtain the open reading frame (ORF) of the AFP, a pair of primers were designed according to the assembled cDNA sequence. The PCR product was then cloned into pMD18-T cloning vector and sequenced. The result showed that the three kinds of insect AFP gene family were obtained. Finally about 19 of MpAFP genes, 5 of ApAFP genes, and 12 of PoAFP genes were cloned from those three kinds of gene family, respectively. Sequence similarity search revealed more than 65% identity at gene level and more than 65% identity at amino acid level with AFPs both from bettle Tenebrio molitor and bettle Dendroides Canadensis. To express the MpAFP in prokaryotic system, three MpAFP genes with different sequences were constructed into the pGEX-4T-1 vectors, respectively, and the recombinants were identified by enzyme restriction digestion and sequencing. The recombinants were then transformed into E.coli BL21(DE3) and induced by IPTG for the expression. The optimal expression conditions were worked out in considering the concentrations of IPTG and the temperatures of bacteria culturing. Based on the optimum conditions, the GST-MpAFPs were expressed at a large scale and furtherly purified through Glutathione Sepharose 4B. At the same time, MpAFP5, the gene without signal peptide sequence, was cloned into pMAL-p2xexpression vector and expressed in E.coli TB1 for preparing the antigen for the following immunological test. The results revealed that the antifreeze protein was expressed in a fusion protein of 64 kDa .It was then purified successfully. To prepare the specific antiserum against MpAFP for the further studies, mice were immunized with GST-MpAFP5 fusion protein, and the produced AFP antiserum could specifically reacted with AFP fusion protein tested by Western blot with MBP-MpAFP5 fusion protein as the antigne. This result furtherly confirmed that the antifreeze protein was expressed correctly. On the other hand, for the further identification of the antiserum and for the high production of antifreeze protein with higher biological activety, three MpAFP genes with different sequences were constructed into pGAPZαA vector, the yeast expression plasmid. Then the recombinants were transformed into yeast Pichia pastoris (SMD1168) after they were identified and linearized.. In the yeast expression system, the protein was expressed out of the cells, so the supernatant of the culture was analyzed by SDS-PAGE and Western blot. The result showed that MpAFP had successfully expressed in yeast cells and secreted into the supernatant. In addition, the biological activity of the expressed antifreeze proteins both in prokaryotic and eukaryotic expression systems was analyzed. The results indicated that the insect antifreeze proteins could protect bacteria from the low temperature damage, and the protective effect was enhanced with the increase of the concentrations of AFP. After the confirmation of its biological function, the MpAFP gene was then constructed into pBI121, a binary vector for the further study of transgenic plants. Taken together, all these results suggested that the insect AFPs we cloned and expressed had biological activities, and these studies also established a solid foundation for better understanding the fuction of insect AFPs and for applying the genes to confer cold tolerance for plants .