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HEAT INDUCIBLE EXPRESSION of ANTIFREEZE PROTEIN GENES from the BEETLES Tenebrio Molitor and Microdera Punctipennis

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HEAT INDUCIBLE EXPRESSION of ANTIFREEZE PROTEIN GENES from the BEETLES Tenebrio Molitor and Microdera Punctipennis CryoLetters 37 (1), 10-18 (2016) © CryoLetters, [email protected] HEAT INDUCIBLE EXPRESSION OF ANTIFREEZE PROTEIN GENES FROM THE BEETLES Tenebrio molitor AND Microdera punctipennis Jieqiong Li, Wenjing Ma and Ji Ma* Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, 14 Shengli Road, 830046 Urumqi, China *Corresponding author e-mail: [email protected] Abstract BACKGROUND: Antifreeze proteins (AFPs) play important roles in protecting poikilothermic organisms from cold damage. The expression of AFP genes (afps) is induced by low temperature. However, it is reported that heat can influence the expression of afps in the desert beetle Microdera punctipennis. OBJECTIVE: To further detect whether heat also induce the expression of afps in other insects, and to determine the expression profiling of insect afps at different temperatures. MATERIALS AND METHODS: The expression of antifreeze protein genes in the two beetles, Microdera punctipennis and Tenebrio molitor that have quite different living environment, under different temperatures were studied by using real–time quantitative PCR. RESULTS: Mild low temperatures (5℃~15℃), high temperature (38℃~47℃ for M. punctipennis, or 37℃~42℃ for T. molitor) and temperature difference (10℃~30℃) all stimulated strongly to the expression of AFP genes (Mpafps) in M. punctipennis which lives in the wild filed in desert. The mRNA level of Mpafps after M. punctipennis were exposed to these temperatures for 1h~5h was at least 30-fold of the control at 25℃. For T. molitor which is breeding in door with wheat bran all these temperatures stimulated significantly to the expression of Tmafps, while the extent and degree of the temperature stimulation on Tmafps expression were much lower than on Mpafps. After T. molitor were exposed to 5℃ and 15℃ for 1h~5h, the mRNA level of Tmafps was over 6-fold and 45-fold of the control at 25℃. High temperature (37℃~42℃) for 1h~3h treatments increased Tmafps mRNA level 4.8-fold of the control. Temperature difference of 10℃ was effective in stimulating Tmafps expression. CONCLUSION: The expression of insect antifreeze protein genes both in M. punctipennis and T. molitor was induced by heat, suggesting that this phenomenon may be common in insects; the extent and degree of the influence differ in species that have different living conditions. The heat inducible expression of antifreeze protein genes hints that antifreeze proteins may involve in other functions except for antifreeze. Keywords: desert beetle; Microdera punctipennis; Tenebrio molitor; antifreeze protein; heat 10 INTRODUCTION test whether the heat inducible expression of insect afps is common in other beetles. The Insects survive cold environment mainly by multi-factor inductivity of the expression of producing antifreeze proteins (AFPs), which can insect antifreeze proteins suggests that antifreeze lower the non-colligative freezing point of water protein may confer insect environmental without depressing the melting point, thus adaptability. The microhabitat of an insect may generate a temperature difference between the strongly influence its behavior due to the usually hysteretic freezing point and the equilibrium small body size and the ectotherm nature of melting point, termed thermal hysteresis activity insect. This may be reflected by the sequence (THA) (3). Antifreeze protein was firstly variability of antifreeze proteins of the insects discovered in Antarctic marine teleost fishes in that have different microhabitats (17, 33), and by 1971 (4), since then, AFPs have been described the differential expression of antifreeze protein in diverse taxa including terrestrial arthropods genes in these insects under the same conditions. (6, 11, 21), bacteria and fungi (8, 32) and plants In this study, two tenebrionids, Microdera (14, 15). The structure of beetle AFP is punctipennis and Tenebrio Molitor, that have characterized by the most regular -helical quite different living habitat were determined by structure composed with different numbers of real time quantitative PCR for the expression repeat unit TCTXSXXCXXAX (where, X pattern of antifreeze protein genes under represents for any amino acids) (19). different temperatures. Both of them produce In general, the expression of antifreeze numerous isoforms of antifreeze protein genes proteins corresponds to the yearly temperature (13, 17, 18, 20, 24, 26). The common yellow changes (1-2, 9-10, 15-16), in that the mealworm T. molitor is a pest of stored grain expression level of antifreeze proteins began to products in cold temperate regions. The survival increase in early autumn, with peaks in winter, of T. molitor in winter is facilitated by and a decline in spring. This yearly expression accumulation of antifreeze proteins (27, 28). T. pattern of antifreeze proteins is consistent with molitor has a relatively stable microhabitat in its function of protecting organisms from cold terms of temperature, humidity and light length damage. However, apparent expression of afps due to living in door. It is relatively easy to and AFPs in summer were also found in beetles maintain cultures of them under laboratory Dendroides canadensis (1), Tenebrio molitor conditions (25˚, 60-70% RH, 16:8 L:D cycle). M. (22), Choristoneura fumiferana (5), Rhagium punctipennis is an endemic species found in mordax (30) and Microdera punctipennis (16, Gurbantonggut desert, northwest of China, 25), though the thermal hysteresis value was low where the climate is characterized by very rare in summer insects (7). Numerous cDNAs of afps rainfall, large temperature difference between have been cloned from M. punctipennis in day and night, and extremely hot in summer. M. summer beetles (17). Most of these sequences punctipennis has well adapted to the harsh desert have one or two of the repeat unit shorter than environment (29) and overwinters in adult (16). those cloned from the overwinter ones, and the In this study we aimed at discovering (1) the amino acid sequences at the C-terminal ends are heat inductivity of the expression of antifreeze also different from each other. protein genes from these two beetle species; (2) In addition to low temperature, several the influence of different temperatures on the environmental factors could induce the expression of antifreeze protein genes to each expression of antifreeze protein genes, such as beetle. Our results showed that the mRNA levels dry and hungry (12), and even heavy metal (22), of the afps from the two species were which suggests that insect antifreeze proteins significantly increased in response to low and may possess other functions except for high temperatures and to temperature antifreeze (1, 24, 25). Interestingly, it was found differences. The effective temperatures for each that the expression of antifreeze protein genes in of them in stimulating the expression of afps the desert beetle M. punctipennis was induced were quite different, suggesting that antifreeze by high temperature, which strongly suggested proteins may confer insects of different that insect antifreeze proteins have other microhabitat different environmental functions besides antifreeze, such as thermal adaptability. Our results will help to further protection (24, 25). At a glance, it is hard to extend the current knowledge on insect predict that the expression of antifreeze protein antifreeze proteins. genes could be induced by heat. So it is worth to 11 MATERIALS AND METHODS refrigerator for RNA extraction. Insect collection Extraction of total RNA and cDNA synthesis M. punctipennis adults were collected from The frozen insect was homogenized in Fukang (N 44˚24, E 087˚51’, 444 m), which is liquid nitrogen in a pre-cold mortar by mixing about 100km north of the geographical center of with 1ml TRIzol reagent (Invitrogen, CA, USA). Asia, and is located at the southern edge of RNA extraction was performed according to the Gurbantonggut Desert in Xinjiang, the northwest manufacturer’s instructions. Each sample was of China. Part of the freshly field-collected treated at 37˚ for 25min with 10 units of RNase- adults were immediately kept in liquid nitrogen, free DNase I (TaKaRa, China) to eliminate any and later were transferred into -80° refrigerator contamination of genomic DNA, then total RNA for latter RNA extraction; the others were was combined with diethyl pyrocarbonate carried back to laboratory for later research. T. (DEPC)-treated water. RNA concentration was molitor were purchased from Hualing market in quantified and quality-checked by using Urumqi, China. NanoDrop ND1000 spectrophotometry (NanoDrop Technologies, USA). The quality of Insect treatment with different temperatures each RNA sample was checked by gel After one week of rearing in laboratory at electrophoresis prior to cDNA synthesis. Total room temperature (about 25℃) adults were RNA (1.0 µg) was reverse transcribed into cDNA with M-MLV Reverse Transcriptase and randomly chosen for temperature treatment. (1) OligodT-Adaptor Primer (3'-Full RACE Core Mild low temperature treatment: insects were Set, TaKaRa, China) with PCR reaction of 70˚ treated at 5˚, 10˚ and 15˚ for 1h, 3h and 5h, for 10min, 42˚ for 60min, 70˚ for 15min. The respectively. (2) High temperature treatment: cDNA was stored at -20˚ until use. insects were treated at 37˚, 42˚ and 47˚ for 1h, 3h and 5h, respectively; T. molitor adults were Primers designed for real-time quantitative treated at 37˚, 40˚ and 42˚ for 1h, 3h and 5h, PCR respectively. (3) Temperature difference Primers for real-time quantitative PCR treatment: insects were randomly divided into (qRT-PCR) to detect the expression of antifreeze three groups, and all were firstly incubated at protein genes in M. punctipennis (Mpafps) and T. 10˚ for 5h, and then at 20˚, 30˚ and 40˚ molitor (Tmafps) after temperature treatment respectively for 5h to generate 10˚, 20˚ and 30˚ were designed according to the sequences of the temperature differences. Insects at room conserved region of each of them by using temperature about 25℃ without any treatment DNAMAN6.0 software. Due to beetle afps exist in many numbers of isorforms which are were used as control.
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