| Anatolica polita, a beetle in Tenebrionidae, is widely distributed in the desert of Xinjiang. It is able to survive by producing antifreeze proteins (AFPs) in the cold environment or during huge temperature fluctuation.To elucidate the mechanism of Anatolica polita adapted to cold environment, the effect of different developmental stages and low temperature on the mRNA levels of Apafps and the influences of environmental factors and seasonal variation on the AFP activity and mRNA accumulation were studied. We also do the preliminary Study of the natural antifreeze protein isolation from Anatolica polita. The results were as follows.The expression of antifreeze protein genes in the immature stages was studied by Real-time PCR. The results showed that mRNA levels of Apafp752 and Apafp914 in the 8th and 9th larvae were the highest which were 8-7 folds of the small instars, but decreased in pupa. The osmolality displayed similar trends with the larva development. This result also implied that development stage is a primary factor in the regulation of expression of Apafps. Under cold stress, Apafps expression in 4-6 instars was significantly increased, indicating that expression of larval Apafps could be stimulated by cold stress. The result of survive rates shows under low temperature, 64% of all 1-3 instars larvae are survived after 12h, but survive rates of 4-6 instars larvae, 7-9 instars larvae and pupae are 77.8%,100% and 100%. Old larvae might be one of the overwintering states in Anatolica polita based on the expression pattern of the immature stages, the survival rate of the larvae under cold stress and field observation.The expression of the two Apafps was high in cold seasons and low in warm seasons. However, there were significant expression of the two antifreeze genes in the hottest months June and July compared to the mRNA level in May for Apafp752, and in August for Apafp914, respectively. In addition, the expression of Apafp752 was higher than Apafp914 throughout the year. The huge difference appeared in early spring, Apafp752 was 3.5 fold of the Apafp914 on the linear scale. Under cold stress both Apafp752 and Apafp914 were up-regulated by low temperature on daily basis (for Apafp752, F5, 24=5.12, P=0.0025; for Apafp914, F5, 18=9.8, P<0.0001). On day 3 after cold treatment the mRNA level of the two genes were both significantly increased than the control sample, and reached peak after 9 and 12 days of cold acclimation, respectively. The response of Apafp752 to the low temperature was stronger than Apafp914.Apafp752 very actively responded to the temperature fluctuation with 5.2 fold and 7.7 fold of the control group after the first and second cycles. Apafp914 appeared only 2.3 fold and 4.5 fold of the control group after the same treatment. Under more than two fluctuation cycles, Apafp752 and Apafp914 mRNA levels respectively decreased to 84% and 79% of their highest levels. Temperature fluctuation also could significantly increased the haemolymph THA (F4, 10=11.48, P<0.0001). After the second cycle, THA apparently increased. Both THA and mRNA expression level slightly decreased after three more cycles. After the first cycle treatment, the increase of the haemolymph THA was later than that of the mRNA.This implied AFPs may play an important role to protect insect in early spring and late autumn.Dry stress could significantly stimulate the expression of the two antifreeze protein genes (for Apafp752 F3, 8=65.37, P<0.0001; for Apafp914 F3, 12=18.54, P<0.0001) within three days of dry stress. On day 3, the mRNA levels of Apafp752 and Apafp914 were 6 and 4 folds of the untreated ones, respectively. The effect of dry stress on Apafps expression was higher than that of the cold stimulation, in which the mRNA levels of Apafp752 and Apafp914 were only 4.4 and 2.2 fold of the control on day 3. In addition, the difference of the expression level between the two genes of the dry treatment was smaller than that in cold treatment, indicating that Apafp752 and Apafp914 responded to cold and dry stresses differently. Measurements on the haemolymph THA indicated that dry stress stimulated the haemolymph THA (F3,8 =27.13, P=0.0002) on day 3. The THA reached to the maximum after 6 days of dry treatment. The expression levels of Apafp752 and Apafp914 were both significantly increased compared to the control (F4, 10=11.48, P=0.0009; F4, 10=10.31, P=0.0014).Preliminary Study of the natural antifreeze protein isolation from Anatolica polita also has been done in our work. The haemolymph was treated by 70℃for 5min, cation exchange, dialyse and freeze-dried. Then the osmolity had been tested by Micro-Sample Osmometer. Four active components had been isolated. Successfully isolated and purification the native AFPs from Anatolica polita will provide information for the further research on structure characteristics and functions of native antifreeze protein.
|
PDF Full Text Request