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Cloning of a cDNA encoding antifreeze protein in Microdera punctipenis dzunarica (Coleoptera: Tenebrionidae) and its activity assay

ZHAO Gan, MA Ji*, XUE Na, YANG Chang-Geng, ZHUAN Fang-Fang, ZHANG Fu-Chun?(Key Laboratory of Molecular Biology, College of Life Science and Technology, Xinjiang University, Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, Urumqi 830046, China)  
For cloning antifreeze protein (AFP) gene from a desert darkling beetle Microdera punctipenis dzunarica in Xinjiang, the primers were designed according to the core sequence of AFP gene deposited in GenBank, and the cDNA fragment about 294 bp named as MpAFP5 was amplified with the RT-PCR and 3'-RACE technique. Sequence analysis revealed that the cloned cDNA fragment named as MpAFP5 coded the mature peptide of AFP. The full sequence of MpAFP5 (AY821792) was about 363 bp. Compared with the Dendroides canadensis AFP-8 and Tenebrio molitor thermal hysteresis protein (THP) isoform 4-9 genes, it had 68.4% and 71.8% identity in gene level, and 70% and 81% identity in protein level respectively. The MpAFP5 gene was then cloned into prokaryotic plasmid pGEX4T-1 and expressed in E. coli BL21(DE3). SDS-PAGE analysis indicated that the fusion antifreeze protein was expressed in E. coli with a molecular weight of about 37 kD. Western-blot analysis showed that MpAFP5 was expressed correctly. Tests on low temperature protection to bacteria showed that the insect antifreeze protein could protect bacteria from the low temperature damages, and the protective effect was correlated with the antifreeze protein concentration.
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