Expression And Functional Analysis Of P38 Signal Pathway Related Genes In Microdera Punctipennis Under Cold Stress

The members of the mitogen-activated protein kinase(MAPK) family participate in cellular responses to a wide range of extracellular stimuli. One of the four sub-families, the p38 group MAP kinases, plays a vital role in numerous biological processes. Researches have showen that the MAPK path way also plays role in insect to respond to cold stress. Previously we have studied the physiological mechanisms in the desert beetle Microdera punctipennis under cold stress, but the regulation mechanism remains unclear. This paper mainly fouces on the relationship of p38 signaling pathway in response to low temperatures, to illustrate wether the MAPK pathway involved in the cold tolerance mechanisms of Microdera punctipennis.We found that most of the genes involving in p38 signaling pathway were upregulated under low temperature conditions( at 4℃ and-4℃), indicating that p38 signaling pathway play role in the cold response. To further investigate this pathway function at low temperatures, we selected the upregulated gene p38 MAPK as our main research kinase. The c DNA of the p38 gene, Mpp38, from the desert beetle M.punctipennis was cloned. E.coli and yeast expression systems were employed to express the Mpp38 and to detecte the founction of Mpp38 under cold stress. We gotmain results as follows:1. Expression profiling of the genes involving in p38 signaling pathway in response to low temperature in M. punctipennis under low temperatures. The c DNA of the genes(ASK1, MLK, TAK1, MKK3, MKK4, p38 MAPK, MAPKAPK, MNK, MSK1,Rho GDI, Tau, ATF4, ETS, Max, CREB1, p53 from M. punctipennis were cloned to construct the standard plasmids for real-time quantitative PCR(q RT-PCR). Our q RT-PCR results showed that the genes in association with tumor, cancer and other diseases,such as TAK1, MNK, Rho GDI, Tau, ATF4, ETS and Max do not respond to low temperatures, while the genes related to stress, such as ASK1, MLK, MKK3,MKK4, p38 MAPK, MAPKAPK2, MSK1, CREB1 and p53, responded to the low temperatures, indicating that p38 signaling pathway not only involve in the regulation of cell growth and differntiation, disease and other activities, but also involve in the cold regulation of M. punctipennis.2. Heterogeneous expression of Mpp38 in E.coli and assay of antifreeze capacity for E. coli. Prokaryotic expression vector p ET28a-Mpp38 was constructed and transformed into E.coli BL21(DE3). His-Mpp38 fusion protein was induced by IPTG,and expressed in soluble form. Western blotting result showed that the His-Mpp38 fusion protein was correctly expressed. The phosphorylation of His-Mpp38 in E. coli was detected; the result indicated that Mpp38 can be phosphorylated in E.coli. The growth of BL21(p ET28a-Mpp38) was better than BL21(p ET28a) after cold treatment, indicating that Mpp38 protein enhanced the cold tolerance of E.coli cells.3. Heterogeneous expression of Mpp38 in yeast and identification of antifreeze capacity for yeast. p YES-Mpp38 eukaryotic expression vector was constructed, and transformed into S.cerevisiae(INVSCⅠ) and induced by galactose for expression of Mpp38 protein,Western blot results showed that the phosphorylation level of Mpp38 increased as time prolonged, while the phosphorylation level of HOG1 decreased, and than reduced gradually till to zero after 30 h of galactose induction. Thus we speculated that the overexpression of Mpp38 in yeast inhibit the phosphorylation of the endogenous HOG1.By detecting the yeast growth curve at low temperatures we found that INVSCⅠ(p YES2) grow better than INVSCⅠ(p YES2-Mpp38), suggesting that the expression of exogenous Mpp38 gene affect the cold tolerance of yeast in response to low temperature. Overexpression of p38 may inhibite yeast hog1 expression, resulting in the impairing of cells antifreeze capacity. The plate colony growth assay of S.cerevisiae cold resistance showed that with the cold treatment time prolonged, the colony numbers of INVSCⅠ(p YES2-Mpp38) was fewer than INVSCⅠ(p YES2).This result is consistent with the growth curves, confirming that Mpp38 weakens the freezing resistancein of s.cerevisiae.To investigate whether Mpp38 weakened the freezing resistance of S.cerevisiae by inhibiting the production of osmotic protective substances, we detected the contents of small molecular osmotic protective substances, such as trehalose, glycerol and proline, as well as H2O2 in yeast, and found that the contents of these substances in INVSCⅠ(p YES2) were higher than those in INVSCⅠ(p YES2-Mpp38),indicating that Mpp38 impact the accumulation of small osmotic protective substances in yeast in response to low temperature.In order to understand the molecular machnism that Mpp38 inhibit the production of small molecular osmotic protective substances, we examined the expression of cold-related genes in yeast HOG1 pathway. The transcriptional level of hog1, GPDH(glyceraldehyde phosphate synthetase), TPS(Trehalose-6-phosphate synthase)and Ole increased in INVSCⅠ(p YES2) under-10℃ treament, but inhibited in INVSCⅠ(p YES2-Mpp38). However, the expression of PBS2, a just upstream kinase gene of hog1 gne, was not affected. These results were consistent with the results of small molecules contents. The expression of Mpp38 reduced the transcriptional level of some of the tested genes downstream of hog1, resulting in the inhibition of the synthesis of small molecular osmotic protective substances, thereby reducing the antifreeze ability of yeast cells.In conclusion, we identified that most of the genes in p38 pathway were involved in cold response in M. punctipennis. The function of Mpp38 was investigated both in prokaryotic and eukaryotic systems. Mpp38 was expressed and phosphorylated, thus enhancing freezing ability of E.coli. However, in the yeast expression system, the expression of p38 inhibited phosphorylation of the endogenous Hog1, thereby inhibiting the expression of the downstream genes, which lead to the weakening of antifreeze ability of yeast. These results indicating, from the nagtive, that Mpp38 do involving in the cold response pathway. The reason that the expression of Mpp38 inhibited the activity of hog1, while not compensate for the function of hog1 needs to be investigated further.