›› 2008, Vol. 51 ›› Issue (5): 480-485.

• RESEARCH PAPERS • Previous Articles     Next Articles

Cloning and activity analysis of insect antifreeze protein gene from Pterocom a loczyi(Coleoptera: Tenebrionidae)

MA Ji   

  • Online:2010-07-27 Published:2008-05-20


To investigate antifreeze protein gene in the beetles liv ing in Xinjiang Junggar desert, Pterocoma loczyi, a
large size desert beetle, was chosen for antifreeze gene cloning. RT-PCR technique was employed to obtain Pterocoma loczyi antifreeze protein gene. One of the obtained cDNA fragment was named Plafp 743.Sequence analysis showed that Plafp 743 encodes 112 amino acids with the repeated constructive sequence CTX1X2X3X4CX5X6X7X8X9, which is the special motif for some of the known insects antifreeze proteins. Recomb inant plasmid pGEX-4T-1-Plafp743 was transferred into Escherichia coli BL21 and induced by IPTG. The expressive product was analyzed by SDS-PAGE, and the result showed that pGEX-4T-1-Plafp743 was expressed in fusion protein with relative molecul ar mass of 36 kD. The fusion protein, named GST-PLAFP, was purified by Glutathione Sepharose 4B, and then identified by Western blotting with mouse antiserum against antifreeze protein from Dendroides canadensis. Then, GST-PLAFP with different concentrations was added to E. coli culture for various days at -6℃ to check its cryoprotective effect on bacteria survival at subzero temperatures. This cryoprotective experiment showed that the fusion protein GST-PLAFP could protect bacteria from freezing to death at conce ntrations as low as 30 μg/mL. The results suggest that AFP might be a widely adopted strategy for overwintering of desert beetles.

Key words: Pterocoma loczyi, antifreeze p rotein (AFP), cloning, sequence analysis, prokaryotic expression, cryoprotecti ve effect