| Hepialus xiaojinensis Tu. is one of the host insects of the precious Chinese herbal medicine, Cordyceps sinensis which has high nutrition. Nowadays, the production of C. sinensis is in short supply. In order to solve the shortage of resources, artificial cultivation is becoming a big deal. Although a lot of unit trials have been carried out for a long time, problems still exist during the artificial cultivation. The most important issue is the extremely low survival rates of the larvae especially when the adverse environmental conditions come. Temperature is considered as the most important factor that affects the survival rate. Mechanisms for insect used to cope with stressful conditions are mainly focused on two aspects. One is the enhanced of protective enzymes such as superoxide dismutase(SOD) and catalase(CAT) during thermal stress, and another is the up-regulated heat shock protein(HSPs) which are ubiquitous in all living organisms. Therefore, this paper mainly focused on the antioxidant enzymes at the physiology level, and concerned the heat shock protein 70(Hsp70) expressions in mRNA at the molecular level, which is very helpful to illustrate thermal stress mechanism of H. xiaojinensis larva under different magnitude of temperature stress. Moreover, the research may be helpful to improve survival rate in artificial cultivation of C. sinensis. The main research contents are as follows: 1. Effects of temperature stress on the activity of antioxidant enzymes in H. xiaojinensis larva.Many previous studies have showed that heat stress can interfere with the redox balance in organisms, and eventually lead to oxidative stress. In this study, seven stress temperatures(0 °C, 4 °C, 12 °C, 16 °C, 20 °C, 24 °C and 28 °C) and two exposure durations including 4 hours and 12 hours were designed. And 8 °C was served as control temperature at which H. xiaojinensis was reared. The H. xiaojinensis fourth instar larvae were placed in each temperature-time combination under the conditions of stress. Then we investigated the activities of four antioxidant enzymes, including superoxide dismutase(SOD), catalase(CAT), glutathione-S-transferase(GSTs), and peroxidase(POD), the variation of total antioxidant capacity(T-AOC) and the concentrations of malondialdehyde(MDA). The results showed that the temperature stress can cause the changes of antioxidant enzyme activity of H. xiaojinensis larvae. The activity of SOD kept at a high level both in the control and treatment groups, which indicated that SOD might play a great role in the process of removing the surplus reactive oxygen species reactive. The activity of CAT in the control group was significantly lower than that of the temperature stress groups. The temperature stress induced the activity of CAT. The activity of POD increased significantly after different temperature treatment stress. And when the activity of CAT decreased after 12 hours stress, the activity of POD increased. This result showed that POD and CAT control the concentration of H2O2 in perfect union. The activity of GSTs after each temperature treatment group was lower than that of the control group. After 12 hours stress, the activity of GSTs showed a decreasing trend. MDA, as an index of lipid peroxidation, had an increasing trend after 24 °C stress. The results of T-AOC showed that the activity of T-AOC was increased significantly after temperature stress to maintain oxidation reduction equilibrium state. 2. Effects of temperature stress on the mRNA expression of Hsp70 in the fat body of H. xiaojinensis larva.The mRNA expression of Hsp70 was measured in the fat body of H. xiaojinensis larva under different temperature stress. With ?-actin as the reference gene, cDNA as the templates, the real-time fluorescent quantitative PCR was constructed to examine the expression levels of Hsp70 in the fat body of H. xiaojinensis larva after temperature stress. The stress temperatures included 16 °C, 24 °C, 28 °C and 32 °C. The durations of each target treatment temperature was 4 hours and 12 hours. The result showed that the high temperature could induce the significantly increased Hsp70 expression(P < 0.05). The expression of Hsp70 after the shorter exposure duration of 4 hours was higher than that of the longer exposure duration 12 hours(P < 0.05). 3. The heat shock proteins of H. xiaojinensis larva after the temperature stress based on the protein group sequencedHeat shock protein family has the function of the molecular chaperone. Molecular chaperone is a special kind of protein molecules which refers to the nascent peptide chain assembly, protein folding and oligomeric protein trans-membrane transport. The stress temperatures were 0 °C, 5 °C, 15 °C, 25 °C and 35 °C, and the exposure duration was 2 hours. The samples were sent to a biotechnology company to be sequenced and analyzed. The COG annotation results showed that the proteins in the H. xiaojinensis of Hsp60 family, Hsp90 family and Hsp10 were in play to the role of the molecular chaperone. |
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