| Microdera punctipennisnnis(Tenebrionidae) is an endemic species in Gurbantonggut Desert in Xinjiang. It is freeze-avoiding. A c-Jun N-terminal kinase(JNK) gene was found to have an up-regulated expression at low temperature in the transcriptomic data of M. punctipennis. Therefore, it is necessary to explore whether this kinase also has the functions at low temperature, except for its immune function.In this study, a c DNA of the c-Jun N-terminal kinase gene from the desert beetle M. punctipennis was cloned(named Mp JNK). Real-time quantitative PCR was performed to analyze the expression profiling of it and the genes involving in JNK signal pathway at low temperatures. E.coli and yeast expression systems were employed to express the Mp JNK and test the function that it enhanced the antifreeze capacity of bacteriar. The yeast system was also employed to test its function at low temperatures. Real-time quantitative PCR(q RT-PCR) was performed to analyze the expression profiling of them at low temperatures. Specific results are as follows:1. Cloning and low temperature expression profiling of Mp JNK. The c DNA of Mp JNK was cloned. Bioinformatic analysis showed that the JNK c DNA from M. punctipennis is 3413 bp long, containing an ORF of 1179 bp, 5’-UTR of 593 bp and 3’-UTR of 1640 bp. The ORF encodes a polypeptide of 392 amino acids. The encoded protein belongs to PKc like super family. Homology analysis showed that Mp JNK shared 97% of identity with the JNK from of Tribolium castaneum. It contains the conserved region of 13 amino acid(IIHRDLKPSNIVV), which is the signature of Serine/threonine protein kinase activation site, indicating that Mp JNK contains an activation site. The ATP binding site and activation loop(166-188) has a TPY(Thr-Pro-Tyr) motif. q RT-PCR results showed that the expression of Mp JNK presented a stress-responsive tendency when stressed at 4℃ and-4℃, that is increasing first, then decreasing. These results suggested that Mp JNK is a low temperature responsive gene which may function in M. punctipennis for dealing with cold stress.2. Detection of the expression profiling of the genes in JNK signaling pathway at low temperature. q-RTPCR results showed that after the beetles were treated at 4℃ and-4℃ stress 0.3~11h, respectively, the expression of the genes which may located at the cell membrane increased at different extent. The relative m RNA level of these cell membrane genes was multiple times of the MAPKKK(including MLK, TAK, POSH and MEKK), MAPKK(including MKK4 and LZK) and MAPK(including JNK, STAT, SMAD and NFAT). In conclusion, the expression magnitude of the genes with locations from cell membrane to cytoplasm and nucleus involving in JNK signal transduction pathway in response to cold stress showed a decreasing tendency.3. Heterogeneous expression of Mp JNK in E.coli and identification of its function in enhancing E.coli freeze tolerance. Mp JNK was inserted into p ET28 a vector and then transformed with E.coli BL21(DE3). The positive clone of BL21(p ET28a-Mp JNK) was selected and induced for the expression of soluble His-Mp JNK. The result of Western blotting showed that the fusion protein His-Mp JNK was correctly expressed. The colony density of the experimental bacteria is greater than the control bacteria after-18℃ treatment, indicating that the heterogeneous expressed Mp JNK conferred BL21(p ET28a-Mp JNK) cells antifreeze resistance.4. Heterogeneous expression of Mp JNK in yeast and identification of its antifreeze function. Mp JNK was inserted into p YES vector and then transformed with yeast strain INVSC I. The positive clone of INVSCⅠ(p YES2-Mp JNK) was selected and used as experimental yeast. The transformed yeast with p YES2 was used as the control. The expression of Mp JNK was induced by galactose. Polyclonal antiserum of Mp JNK was prepared in mouse by using DNA immunization with pc DNA3-Mp JNK plasmid.After treated at-18℃, the growing curves of the yeast INVSCⅠ(p YES2-Mp JNK) and INVSCⅠ(p YES2) cells recovery at 37℃ for periods of time were fitted, the results showed that the control yeast growth better than the experimental yeast. After 5d of cold stress the experimental yeast stopped growing, indicating that the heterogeneous expressed Mp JNK inhibited the cold resistance of the yeast cells. Detection of experimental INVSC I( p YES2-Mp JNK) and the control INVSC I(p YES2) the stress resistance index under low temperature stress, proline, soluble sugar and glycerol content, the results showed that the control yeast growth better than the experimental yeast. Indicating that the insertion of exogenous gene inhibit the synthesis of physiological indexes of stress resistance.We prepared a polyclonal antibody of Mp JNK in mouse by using DNA immunization with pc DNA3-Mp JNK plasmid. The titer of the mouse anti-Mp JNK antiserum was up to 1:12 800. Western blotting result showed that this antiserum had a specific binding to the lysate of INVScⅠ(p YES2-Mp JNK), indicating that Mp JNK was correctly expressed in INVScⅠ.5. Expression profiling of the genes evolving in HOG signal transduction pathways, a homologous of the JNK signaling pathway, at low temperatures. INVSCⅠ(p YES2-Mp JNK) and INVSCⅠ(p YES2) cells were treated under 4℃ and-18℃ from 5min to 8d. q RT-PCR results showed that the expression of the genes in HOG signal transduction pathways, from INVScⅠ(p YES2-Mp JNK, presented a different degree of increase within 5min~60min, while from 1~8d cold treatment the expression presented a significant down regulation compared with that of the INVSC I(p YES2), indicating that HOG signal transduction pathways related genes in S. cerevisiae was inhibited by the heterogeneous Mp JNK.Through research we found, JNK(c-Jun amino terminal kinase) as a member of MAPK pathway family. Not only activation of downstream nuclear transcription factor, mediated organs development change of cells, through the transcription and untranscription dependence form, but also built it on different vector like E. coli and S. crevisiae it can response to low temperature stress. |
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