冷胁迫条件下小胸鳖甲p38信号转导通路相关基因的表达及功能
发布时间：2018-09-06 13:13
【摘要】：丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)家族在响应胞外刺激时调控细胞的响应过程。p38作为该家族的成员之一,在真核生物应激响应过程中发挥重要作用。已有研究表明MAPK通路参与了昆虫耐寒机制的调控,本实验室虽然对拟步甲科荒漠昆虫小胸鳖甲(Microdera punctipennis)的低温生理机制已有大量研究,但对于小胸鳖甲的低温调控机制还不清楚。本文主要研究了小胸鳖甲p38信号通路与响应低温的关系,旨在说明MAPK通路是否参与了小胸鳖甲的抗冻机制。通过检测p38信号通路相关基因的低温表达谱发现该通路的大部分基因在低温条件下(4℃)上调表达,为了进一步研究该通路的主要因子在低温条件下的功能,选择上调表达的p38MAPK基因作为研究对象,以小胸鳖甲c DNA为模板扩增了p38的全长序列,利用大肠杆菌和酵母表达系统,对小胸鳖甲p38蛋白(Mpp38)的抗冻功能进行研究,具体内容及结果如下:1.小胸鳖甲p38信号通路相关基因低温表达谱分析。通过检测小胸鳖甲p38信号通路中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)等基因的低温表达谱,我们发现除了与肿瘤、癌症等疾病相关的基因(TAK1,MNK,Rho GDI,Tau,ATF4,ETS,Max)不响应低温外,大部分与应激相关的基因(ASK1,MLK,MKK3,MKK4,p38MAPK,MAPKAPK2,MSK1,CREB1,p53)都响应低温而上调表达,说明p38信号通路可能参与了小胸鳖甲应对低温胁迫的调控机制。2.Mpp38的原核表达与增强细菌抗冻性分析。构建p ET28a-Mpp38表达载体,并转化大肠杆菌BL21(DE3),经IPTG诱导表达,获得His-Mpp38融合蛋白。Western blot鉴定融合蛋白的正确表达。检测确认p38的磷酸化水平,说明Mpp38在细菌内能被磷酸化。通过低温(-10℃)处理转基因大肠杆菌的生长曲线发现BL21(p ET28a-Mpp38)比BL21(p ET28a)长得快,说明Mpp38蛋白在大肠杆菌抗冻过程中发挥作用。3.利用酵母系统对Mpp38蛋白进行功能研究。构建p YES-Mpp38真核表达载体并转化酿酒酵母(INVSCⅠ),经半乳糖诱导表达Mpp38蛋白,Western blot检测诱导不同时间后p38的磷酸化水平变化,发现随着诱导时间的延长,p38的磷酸化水平越来越高,而酵母细胞的p38对应物hog1的磷酸化水平无太大差异,但诱导30h后,hog1的磷酸化水平降低,甚至完全受到抑制。由此我们推测,小胸鳖甲p38在酵母细胞的外源过表达抑制了酿酒酵母内源hog1的磷酸化。通过对低温条件下酵母生长曲线检测发现INVSCⅠ(p YES2)的生长明显比INVSCⅠ(p YES2-Mpp38)快,说明外源Mpp38蛋白的表达影响了酵母响应低温时的正常生长。可能是Mpp38的过表达抑制了酵母hog1的表达,导致INVSCⅠ(p YES2-Mpp38)抗冻能力减弱。从酵母的滴板实验中发现,随着低温处理的时间的延长,INVSCⅠ(p YES2)和INVSCⅠ(p YES2-Mpp38)的菌落数越来越少,但INVSCⅠ(p YES2)的菌落数比INVSCⅠ(p YES2-Mpp38)的多,也说明了p38在酵母中的表达减弱了酿酒酵母的抗冻性,与生长曲线结果一致。为了进一步验证Mpp38对酿酒酵母抗冻的影响,我们又检测了转基因酵母的小分子渗透保护物质(海藻糖、甘油、脯氨酸)以及H2O2的含量,发现INVSCⅠ(p YES2)藻糖、甘油、脯氨酸物质含量都比INVSCⅠ(p YES2-Mpp38)高;说明Mpp38的表达降低了酵母响应低温时小分子渗透保护物质的积累。为了深入了解Mpp38对酵母hog1的抑制所带来的影响,我们检测了酵母HOG1通路低温相关基因的低温表达谱。-10℃促进INVSCⅠ(p YES2)中高渗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、水杨酸甲酯转移酶Ole等基因的表达,却抑制了INVSCⅠ(p YES2-Mpp38)中这些基因的表达,而对hog1上游基因PBS2(MAPKK)无影响。这些基因的表达情况与小分子物质含量的结果一致。说明Mpp38的过表达抑制了HOG1的活性,导致低温条件下hog1下游的基因表达受影响、小分子物质合成减少,进而降低酵母的抗冻能力。综上所述,通过检测小胸鳖甲p38信号通路中的16个基因在低温下的m RNA水平,确定p38MAPK信号通路参与小胸鳖甲对低温的响应。通过大肠杆菌与酵母系统验证p38MAPK基因的功能,证明在原核系统中,Mpp38可被大肠杆菌细胞磷酸化,并且提高了低温下大肠杆菌细胞内抗冻物质的积累,从而提高了细菌的抗冻能力。在酵母表达系统中,p38的表达抑制了酵母内源p38同源物hog1的磷酸化,阻断了酵母自身的HOG1通路,导致转基因酵母抗冻能力减弱。这一结果从反面证明小胸鳖甲p38通路参与细胞低温响应。但是p38作为hog1的同源基因,抑制hog1活性后却不能弥补其功能的原因有待进一步研究。
[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.
【学位授予单位】：新疆大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q963
本文编号：2226455
[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.
【学位授予单位】：新疆大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q963
【相似文献】
相关期刊论文 前4条
1 孟闪闪;马文静;刘小宁;马纪;;β-actin基因在荒漠昆虫光滑鳖甲和小胸鳖甲中表达的稳定性研究[J];天津农业科学;2014年02期
2 石萌;刘晓静;刘小宁;马纪;;温度胁迫对荒漠昆虫小胸鳖甲(鞘翅目:拟步甲科)幼虫热激蛋白基因hsp70表达的影响[J];环境昆虫学报;2014年03期
3 陈亮;张富春;黄萍;马纪;;光滑鳖甲热休克蛋白70基因的克隆及表达[J];昆虫学报;2007年09期
4 ;[J];;年期
相关会议论文 前4条
1 邢延一;雷海民;;炙鳖甲中总氨基酸含量测定[A];中华中医药学会第四届中药炮制分会学术会议论文集[C];2004年
2 刘清池;马传宝;李建英;庞宇慧;郭丽改;熊建军;王荣孝;张慧敏;张振会;谢勇;梁春耕;冯新旺;武大勇;吴维海;;鳖甲生血丸治疗原发性骨髓纤维化120例疗效观察[A];中华中医药学会第二届岐黄论坛——血液病中医药防治分论坛论文集[C];2014年
3 李力强;黄胜英;宾建平;李明;翟洪;卢国清;周海忠;;鳖甲消症丸预防肝硬变患者食道胃底静脉曲张破裂出血的临床研究[A];第九次全国中西医结合肝病学术会议论文集[C];2000年
4 刘彦;刘承初;;鳖甲与龟甲比较研究[A];2010年中国农业工程学会农产品加工及贮藏工程分会学术年会暨华南地区农产品加工产学研研讨会论文摘要集[C];2010年
相关重要报纸文章 前10条
1 赵春光;鳖甲药用价值高[N];中国渔业报;2004年
2 韩德承;识别真假鳖甲[N];中国中医药报;2013年
3 韩德承;鳖与鳖甲的药用[N];上海中医药报;2007年
4 罗加;鳖甲与光绪帝的腰痛[N];中国信息报;2002年
5 常怡勇;鳖甲神效医腰痛[N];中国中医药报;2006年
6 甘肃省酒泉市中医院 王世彪邋何继红;巧用鳖甲治腰痛[N];中国中医药报;2008年
7 赵民生;龟甲与鳖甲的异同[N];中国中医药报;2010年
8 梁庆森;煎煮过的龟板鳖甲不宜药用[N];中国中医药报;2002年
9 李佩文;甲鱼大补别乱吃[N];健康报;2006年
10 农训学;鳖甲的加工[N];中国中医药报;2006年
相关博士学位论文 前1条
1 高建蓉;鳖甲防治肝纤维化及其作用机制的实验研究[D];湖北中医学院;2007年
相关硕士学位论文 前8条
1 孟闪闪;冷胁迫条件下小胸鳖甲p38信号转导通路相关基因的表达及功能[D];新疆大学;2016年
2 施婧妮;鳖甲炮制前后抗肝纤维化药效物质及其质量分析比较研究[D];湖北中医药大学;2011年
3 韩秋俊;基于鳖甲功效的先导化合物的发现研究（二）[D];北京中医药大学;2013年
4 邢延一;鳖甲中寡肽类化学成分及炮制原理的研究[D];北京中医药大学;2006年
5 陈进文;鳖甲抗肝纤维化活性物质及其指纹图谱研究[D];湖北中医学院;2009年
6 孔菲菲;鳖甲活性多肽的制备工艺及其抗肝纤维化的药效学研究[D];湖北中医药大学;2014年
7 王岩;准噶尔小胸鳖甲和光滑鳖甲的生物学特性及其人工饲养体系的研究[D];新疆大学;2010年
8 张坦;微粉化鳖甲山楂颗粒剂的药学研究[D];湖北中医学院;2005年
，本文编号：2226455
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2226455.html
最近更新
教材专著
