Expression Analysis Of Heat Shock Protein 70 Gene And Antifreeze Protein Gene From Anatolica Polita Borealis

The weather in Xinjiang desert region is a kind of typical dry land weather. The temperature fluctuations are greatly during the year and extreamly in one day. There are abundant insects living in the desert region of Xinjiang. Tenebrionidae is a class of typical insects possessing the specific mechanism of adaptation and resistance to desert weather in Xinjiang. They successfully survive and reproduce in desert after long time evolution. The extremely drought and temperature fluctuations make it an ideal place for study the mechnisam of insects adaptation and resistance to adverse conditions, such as high temperature, low temperature and drought.In our study, Anatolica polita borealis was selected as the experimental material which is one of the broadly distributed organisms in desert region. Anatolica polita borealis has much close relationship with desert environment, which, to some certain extent, is linked with heat shock proteins 70 (HSP70s) and antifreeze proteins (AFPs). So it is of great significance to research the function of HSP70s and AFPs in desert acclimation and cold tolerance.Heat shock protein 70 (HSP70s) is the most important proteins among members of heat shock proteins family and functions mainly as molecular chaperones. Moreover, it plays important roles in cellular protection, anti-apoptotic effects, and immune therapy for tumor. Therefore, researches about HSP70s have been one of important and prospective focuses in fields of life sciences. Antifreeze proteins designate a class of proteins that are able to bind to ice and inhibit the generation and growth of ice crystal. AFPs can depress the freezeing point while not change the melting point of solution, and they also can maintain the stability of super cooling state of fluid.The main tasks of this work are composed of three parts including: 1.cloning and sequencing of heat shock protein 70 gene from Anatolica polita borealis; 2. Expression analysis of heat shock protein 70 gene in Anatolica polita borealis; 3. Expression analysis of antifreeze protein gene in Anatolica polita borealis.1. Cloning and sequencing of heat shock protein 70 gene from Anatolica polita borealisThe hsp70 gene from Anatolica polita borealis was cloned through PCR and RACE technique. Sequence analysis indicated that the APhsp70 gene has 2092 bp in length, includes one full open reading fragment and potentially encodes 653 amino acid residues. The protein deduced from the isolated gene had a calculated molecular mass of 70 kDa and was therefore named APHSP70, and shared high identity with the HSP70 of Tribolium castaneum (red flour beetle) (94.18%). The APhsp70 gene does not display any intron. In general, this is the first time to clone the gene encoding heat shock protein in Anatolica polita borealis.2. Expression analysis of heat shock protein 70 gene in Anatolica polita borealisThe expression pattern of APhsp70 was examined at the transcription level after treated with heat and cold stress. The Northern blot and Semi-quantitative RT-PCR results revealed that the APHSP70 was an inducible 70 kDa isoform whose expression is specifically induced by the different stress factors.3. Expression analysis of antifreeze protein gene in Anatolica polita borealis Antifreeze proteins (AFPs), found in certain organisms enduring freezing environments, have the ability to inhibit damaging ice crystal growth. In this study, the copy number of the cloned gene Apafp914 was estimated by Southern blot technique. The expression pattern of Apafp914 was examined at the transcription level after cold hardening. The Semi-quantitive RT-PCR results revealed that the Apafp914 was up-regulated after long cold hardening. Through calculated Integrated Density Value of electrophoresis image of the semi-quantitive RT-PCR product, the difference between normal group and treated five-day group was significant (P< 0.05 ). But there was no obviours change after rapid cold hardening.