洛氏脊漠甲抗冻蛋白基因的克隆、表达和功能检测

Abstract

Abstract:

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

MA Ji. Cloning and activity analysis of insect antifreeze protein gene from Pterocom a loczyi(Coleoptera: Tenebrionidae)[J]., 2008, 51(5): 480-485.

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

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