發(fā)布時間：2018-09-06 13:13

【摘要】：絲裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)家族在響應(yīng)胞外刺激時調(diào)控細(xì)胞的響應(yīng)過程。p38作為該家族的成員之一,在真核生物應(yīng)激響應(yīng)過程中發(fā)揮重要作用。已有研究表明MAPK通路參與了昆蟲耐寒機(jī)制的調(diào)控,本實驗室雖然對擬步甲科荒漠昆蟲小胸鱉甲(Microdera punctipennis)的低溫生理機(jī)制已有大量研究,但對于小胸鱉甲的低溫調(diào)控機(jī)制還不清楚。本文主要研究了小胸鱉甲p38信號通路與響應(yīng)低溫的關(guān)系,旨在說明MAPK通路是否參與了小胸鱉甲的抗凍機(jī)制。通過檢測p38信號通路相關(guān)基因的低溫表達(dá)譜發(fā)現(xiàn)該通路的大部分基因在低溫條件下(4℃)上調(diào)表達(dá),為了進(jìn)一步研究該通路的主要因子在低溫條件下的功能,選擇上調(diào)表達(dá)的p38MAPK基因作為研究對象,以小胸鱉甲c DNA為模板擴(kuò)增了p38的全長序列,利用大腸桿菌和酵母表達(dá)系統(tǒng),對小胸鱉甲p38蛋白(Mpp38)的抗凍功能進(jìn)行研究,具體內(nèi)容及結(jié)果如下:1.小胸鱉甲p38信號通路相關(guān)基因低溫表達(dá)譜分析。通過檢測小胸鱉甲p38信號通路中MKKK(ASK1,MLK,TAK1,),MKK(MKK3,MKK4),MAPK(p38MAPK,MNK),下游激酶(MAPKAPK,MSK1),下游底物(Rho GDI,Tau,ATF4,ETS,Max,CREB1,p53)等基因的低溫表達(dá)譜,我們發(fā)現(xiàn)除了與腫瘤、癌癥等疾病相關(guān)的基因(TAK1,MNK,Rho GDI,Tau,ATF4,ETS,Max)不響應(yīng)低溫外,大部分與應(yīng)激相關(guān)的基因(ASK1,MLK,MKK3,MKK4,p38MAPK,MAPKAPK2,MSK1,CREB1,p53)都響應(yīng)低溫而上調(diào)表達(dá),說明p38信號通路可能參與了小胸鱉甲應(yīng)對低溫脅迫的調(diào)控機(jī)制。2.Mpp38的原核表達(dá)與增強(qiáng)細(xì)菌抗凍性分析。構(gòu)建p ET28a-Mpp38表達(dá)載體,并轉(zhuǎn)化大腸桿菌BL21(DE3),經(jīng)IPTG誘導(dǎo)表達(dá),獲得His-Mpp38融合蛋白。Western blot鑒定融合蛋白的正確表達(dá)。檢測確認(rèn)p38的磷酸化水平,說明Mpp38在細(xì)菌內(nèi)能被磷酸化。通過低溫(-10℃)處理轉(zhuǎn)基因大腸桿菌的生長曲線發(fā)現(xiàn)BL21(p ET28a-Mpp38)比BL21(p ET28a)長得快,說明Mpp38蛋白在大腸桿菌抗凍過程中發(fā)揮作用。3.利用酵母系統(tǒng)對Mpp38蛋白進(jìn)行功能研究。構(gòu)建p YES-Mpp38真核表達(dá)載體并轉(zhuǎn)化釀酒酵母(INVSCⅠ),經(jīng)半乳糖誘導(dǎo)表達(dá)Mpp38蛋白,Western blot檢測誘導(dǎo)不同時間后p38的磷酸化水平變化,發(fā)現(xiàn)隨著誘導(dǎo)時間的延長,p38的磷酸化水平越來越高,而酵母細(xì)胞的p38對應(yīng)物hog1的磷酸化水平無太大差異,但誘導(dǎo)30h后,hog1的磷酸化水平降低,甚至完全受到抑制。由此我們推測,小胸鱉甲p38在酵母細(xì)胞的外源過表達(dá)抑制了釀酒酵母內(nèi)源hog1的磷酸化。通過對低溫條件下酵母生長曲線檢測發(fā)現(xiàn)INVSCⅠ(p YES2)的生長明顯比INVSCⅠ(p YES2-Mpp38)快,說明外源Mpp38蛋白的表達(dá)影響了酵母響應(yīng)低溫時的正常生長。可能是Mpp38的過表達(dá)抑制了酵母hog1的表達(dá),導(dǎo)致INVSCⅠ(p YES2-Mpp38)抗凍能力減弱。從酵母的滴板實驗中發(fā)現(xiàn),隨著低溫處理的時間的延長,INVSCⅠ(p YES2)和INVSCⅠ(p YES2-Mpp38)的菌落數(shù)越來越少,但I(xiàn)NVSCⅠ(p YES2)的菌落數(shù)比INVSCⅠ(p YES2-Mpp38)的多,也說明了p38在酵母中的表達(dá)減弱了釀酒酵母的抗凍性,與生長曲線結(jié)果一致。為了進(jìn)一步驗證Mpp38對釀酒酵母抗凍的影響,我們又檢測了轉(zhuǎn)基因酵母的小分子滲透保護(hù)物質(zhì)(海藻糖、甘油、脯氨酸)以及H2O2的含量,發(fā)現(xiàn)INVSCⅠ(p YES2)藻糖、甘油、脯氨酸物質(zhì)含量都比INVSCⅠ(p YES2-Mpp38)高;說明Mpp38的表達(dá)降低了酵母響應(yīng)低溫時小分子滲透保護(hù)物質(zhì)的積累。為了深入了解Mpp38對酵母hog1的抑制所帶來的影響,我們檢測了酵母HOG1通路低溫相關(guān)基因的低溫表達(dá)譜。-10℃促進(jìn)INVSCⅠ(p YES2)中高滲透性甘油促分裂原活化蛋白激酶hog1、甘油三磷酸合成酶GPDH、海藻糖-6-磷酸合成酶TPS、水楊酸甲酯轉(zhuǎn)移酶Ole等基因的表達(dá),卻抑制了INVSCⅠ(p YES2-Mpp38)中這些基因的表達(dá),而對hog1上游基因PBS2(MAPKK)無影響。這些基因的表達(dá)情況與小分子物質(zhì)含量的結(jié)果一致。說明Mpp38的過表達(dá)抑制了HOG1的活性,導(dǎo)致低溫條件下hog1下游的基因表達(dá)受影響、小分子物質(zhì)合成減少,進(jìn)而降低酵母的抗凍能力。綜上所述,通過檢測小胸鱉甲p38信號通路中的16個基因在低溫下的m RNA水平,確定p38MAPK信號通路參與小胸鱉甲對低溫的響應(yīng)。通過大腸桿菌與酵母系統(tǒng)驗證p38MAPK基因的功能,證明在原核系統(tǒng)中,Mpp38可被大腸桿菌細(xì)胞磷酸化,并且提高了低溫下大腸桿菌細(xì)胞內(nèi)抗凍物質(zhì)的積累,從而提高了細(xì)菌的抗凍能力。在酵母表達(dá)系統(tǒng)中,p38的表達(dá)抑制了酵母內(nèi)源p38同源物hog1的磷酸化,阻斷了酵母自身的HOG1通路,導(dǎo)致轉(zhuǎn)基因酵母抗凍能力減弱。這一結(jié)果從反面證明小胸鱉甲p38通路參與細(xì)胞低溫響應(yīng)。但是p38作為hog1的同源基因,抑制hog1活性后卻不能彌補(bǔ)其功能的原因有待進(jìn)一步研究。
[Abstract]:The mitogen-activated protein kinase (MAPK) family regulates cell response to extracellular stimuli. As a member of this family, p38 plays an important role in eukaryotic stress response. Studies have shown that MAPK pathway is involved in the regulation of insect cold tolerance, although our laboratory has been able to do so. The physiological mechanism of hypothermia in Microdera punctipennis, a desert insect belonging to the family Carapaceae, has been extensively studied, but the regulation mechanism of hypothermia in Microdera punctipennis is still unclear. In order to further study the function of the main factors of the p38 signaling pathway under low temperature, the p38 MAPK gene was selected as the research object, and the full length of p38 was amplified by using the C DNA template of the small breast turtle shell. The cryoprotective function of p38 protein (Mpp38) was studied by using E. coli and yeast expression system. The specific contents and results were as follows: 1. The cryoprotective expression profiles of genes related to p38 signaling pathway were analyzed. MKKK (ASK1, MLK, TAK1,), MKK (MKK3, MKK4), MAPK (p38MAPK, MNK) in p38 signaling pathway were detected. The low-temperature expression profiles of kinases (MAPKAPK, MSK1), downstream substrates (Rho GDI, Tau, ATF4, ETS, Max, CREB1, p53) showed that most of the stress-related genes (ASK1, MLK, MK3, MK4, p38MAPK, MAPK2, MSK1, CREB1, p53) did not respond to low temperature except for the genes associated with tumors and cancer (TAK1, MNK, Rho GDI, Tau, ATF4, ETS, Max). The up-regulated expression of p38 in response to low temperature indicated that p38 signaling pathway might be involved in the regulation mechanism of hypothermic stress. 2. Prokaryotic expression of Mpp38 and enhancement of bacterial freeze resistance analysis. The expression vector of P ET28a-Mpp38 was constructed and transformed into E. coli BL21 (DE3), which was induced by IPTG to obtain his-Mpp38 fusion protein. Western blot was used to identify the fusion protein. The phosphorylation level of p38 was confirmed, indicating that Mpp38 could be phosphorylated in bacteria. BL21 (p ET28a-Mpp38) grew faster than BL21 (p ET28a) through the growth curve of transgenic E. coli treated at low temperature (-10 C), indicating that Mpp38 played a role in the process of E. coli freezing resistance. 3. Eggs of Mpp38 were treated with yeast system. The expression vector of P YES-Mpp38 was constructed and transformed into Saccharomyces cerevisiae (INVSC I). The expression of Mpp38 protein was induced by galactose. The phosphorylation level of p38 was detected by Western blot. It was found that the phosphorylation level of p38 increased with the prolongation of induction time, while that of p38 corresponding to Hog1 in yeast cells. The phosphorylation level of Hog1 was not significantly different, but the phosphorylation level of Hog1 was decreased or even completely inhibited 30 h after induction. We speculated that the exogenous overexpression of p38 in yeast cells inhibited the phosphorylation of endogenous Hog1 in Saccharomyces cerevisiae. The growth curve of INVSC I (p YES2) was detected under low temperature. The expression of exogenous Mpp38 protein was faster than that of INVSC I (p YES2-Mpp38), suggesting that the expression of exogenous Mpp38 protein affected the normal growth of yeast in response to low temperature. It may be that the overexpression of Mpp38 inhibited the expression of Hog1 in yeast, resulting in the weakening of anti-freezing ability of INVSC I (p YES2-Mpp38). The colony number of INVSC I (p YES2-Mpp38) was less and less, but the colony number of INVSC I (p YES2) was more than that of INVSC I (p YES2-Mpp38). The expression of p38 in Saccharomyces cerevisiae weakened the freeze resistance of Saccharomyces cerevisiae, which was consistent with the growth curve. The contents of trehalose, glycerol, proline and H2O2 were higher in INVSC I (p YES2) than in INVSC I (p YES2-Mpp38), indicating that the expression of Mpp38 decreased the accumulation of osmotic protectants in yeast under low temperature. We examined the expression profiles of genes associated with hypothermia in the yeast HOG1 pathway. -10 C promoted the expression of high osmotic glycerol-activated protein kinase hog1, glycerol triphosphate synthase GPDH, trehalose-6-phosphate synthase TPS, methyl salicylate transferase Ole in INVSC I (p YES2), but inhibited the expression of INVSC I (p The expression of these genes in YES2-Mpp38 had no effect on the upstream gene PBS2 (MAPKK) of hog1. The expression of these genes was consistent with the content of small molecular substances. In summary, the p38 MAPK signaling pathway was involved in the response of Trionyx micropectoralis to hypothermia by detecting the m RNA levels of 16 genes in the p38 signaling pathway. In yeast expression system, the expression of p38 inhibited the phosphorylation of endogenous p38 homologue hog1, blocked the HOG1 pathway of yeast itself, resulting in the weakening of the freezing resistance of transgenic yeast. The p38 pathway is involved in the cell response to hypothermia. However, the reason why p38, as a homologous gene of hog1, can not compensate for its function after inhibiting the activity of Hog1 remains to be further studied.
【學(xué)位授予單位】：新疆大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q963

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