冷胁迫条件下荒漠昆虫小胸鳖甲JNK信号转导通路相关基因的表达及功能研究
发布时间：2019-05-21 07:39
【摘要】：荒漠昆虫小胸鳖甲(Microdera punctipennisnnis)作为新疆准噶尔盆地的拟步甲科的特有种,属避冻型昆虫。在小胸鳖甲低温转录组数据库中分析发现c-Jun N端激酶(c-Jun N-terminal kinase,JNK)基因上调表达,并获得该激酶的相关基因。已知该MAPK JNK信号转导通路参与免疫防御作用,然而该通路在低温环境胁迫时是否发挥作用,值得我们深入研究。本研究以小胸鳖甲的c DNA作为模板,克隆得到MpJNK基因及其相关基因。利用RT-PCR技术分别分析不同时间不同温度的表达谱,利用大肠杆菌和酵母表达系统,验证MpJNK蛋白的抗冻功能,通过将重组质粒转化至酿酒酵母,利用RT-PCR技术检测出MpJNK在酿酒酵母中抑制了其生长,具体结果如下:1.小胸鳖甲c-Jun N端激酶(c-Jun N-terminal kinase,JNK)基因的克隆与低温表达谱。以小胸鳖甲cDNA为模板克隆获得c-Jun N端激酶基因,MpJNK。生物信息学分析表明,小胸鳖甲JNK cDNA全长3413bp,命名为MpJNK,它包含1179bp的开放阅读框、593bp的5’-UTR和1640bp的3’-UTR,编码392个氨基酸,属于PKc like超家族,命名为MpJNK。MpJNK与赤拟谷盗JNK的同源性达97%。该序列在145~157处有保守的13个氨基酸序列(IIHRDLKPSNIVV),为丝氨酸/苏氨酸蛋白激酶激活位点的签名序列,预测小胸鳖甲JNK蛋白包含一个激活位点,ATP结合位点以及活化环(166-188),并含有一个TPY(Thr-Pro-Tyr)的模体。实时荧光定量PCR结果显示,在4℃和-4℃低温胁迫1h后,MpJNK的mRNA水平都显著高于室温对照,并呈现先升高后降低的应激响应趋势,该基因是冷响应基因,对小胸鳖甲应对低温胁迫可能发挥作用。2.JNK信号转导通路相关基因的低温表达谱分析。通过对小胸鳖甲JNK信号转导通路相关基因在不同时间低温胁迫下的相对表达量变化进行检测,实时荧光定量PCR结果显示,4℃以及-4℃胁迫小胸鳖甲成虫0.3~11 h后,MpJNK相关基因的mRNA水平的变化呈现出位于细胞膜上的JNK信号转导通路的相关基因均发生不同程度的上调表达,且相对表达量的倍数高于MAPKKK(包括MLK、TAK、POSH、MEKK),MAPKK(包括MKK4、LZK)以及MAPK(包括JNK、STAT、smad和nfat),即小胸鳖甲jnk信号转导通路相关基因对低温冷响应的表达趋势可概括为从细胞膜到细胞质最终到达细胞核的mrna的相对表达量伴随jnk信号转导通路的级联模式呈逐级递减趋势。3.mpjnk的原核表达及其增强细菌抗冻功能的验证。将mpjnk构建于pet28a载体上,转化大肠杆菌bl21(de3),然后筛选阳性单克隆bl21(pet28a-mpjnk),诱导表达获得可溶性蛋白。利用westernblot印迹技术结果显示,重组蛋白his-mpjnk表达正确。实验菌与对菌在-18℃的生长曲线结果显示,重组的实验菌生长优于对照菌。表明his-mpjnk可能赋予大肠杆菌bl21(pet28a-mpjnk)的抗冻能力。4.采用真核表达系统进行mpjnk蛋白的功能研究。将mpjnk构建至pyes2载体上,转化至酿酒酵母(invscⅠ)中,并获得实验菌invscⅠ(pyes2-mpjnk),对照组为载体pyes2转化菌invscⅠ(pyes2)中,半乳糖用以诱导外源蛋白mpjnk表达。-18℃不同时间低温胁迫实验菌、对照菌生长曲线结果表明,对照菌的生长优于实验菌;仅仅胁迫5d后试验菌便表现出停止生长,该结果表明酵母表达的mpjnk可能抑制了胁迫的耐受性。检测实验菌invscⅠ(pyes2-mpjnk)和对照组菌invscⅠ(pyes2)低温胁迫下的抗逆性指标脯氨酸,可溶性糖及甘油含量,实验结果显示对照菌的抗逆性指标高于实验组。表明外源基因的插入可能抑制了酵母抗逆性生理指标的合成。利用核酸免疫小鼠制备mpjnk蛋白的特异性抗体,获得mpjnk小鼠抗血清,效价为1:12800,以此为一抗进行westernblot,检测invscⅠ(pyes2-mpjnk)超声后的裂解液,酵母外源蛋白在目的条带处正确表达。5.酵母系统中与jnk信号通路同源的hog信号通路相关基因的低温表达谱。利用实时荧光定量pcr对实验菌和对照菌hog信号转导通路相关基因在不同时间低温胁迫下的相对表达量变化进行检测,结果显示4℃和-18℃胁迫重组酵母菌5min至8d,hog相关基因的mrna水平的变化在短时间内(5min~60min)呈现出不同程度的上调表达,然而和对照菌相比较在1~8d时呈现出显著性下调,表明在重组酿酒酵母中,hog信号通路的相关基因的表达被抑制。通过研究我们发现,JNK(c-Jun氨基末端激酶)作为MAPK(丝裂原活化蛋白激酶)家族的一员,不仅能够通过转录和非转录的依赖形式,活化下游核转录因子,介导器官发育期的细胞改变,而且将其构建于不同的载体转化大肠杆菌以及酿酒酵母后也能响应低温的胁迫。
[Abstract]:Microdera punctipennnis, a desert insect, is the endemic species of the quasi-step-in-one family of the Zhilerian basin of Xinjiang, and belongs to the non-freezing-type insects. The expression of c-Jun N-terminal kinase (JNK) in c-Jun N-terminal kinase (JNK) gene was detected in the low-temperature transcriptome database of the small chest and turtle shell, and the related gene of the kinase was obtained. It is known that the MAPK JNK signal transduction pathway is involved in the immune defense, but whether the pathway plays a role in low-temperature environmental stress is worthy of our in-depth study. The MpJNK gene and its related genes were obtained by using c-DNA as a template in the study. The anti-freezing function of the MpJNK protein is verified by using the RT-PCR technology, the anti-freezing function of the MpJNK protein is verified by using the Escherichia coli and the yeast expression system, the recombinant plasmid is transformed into the Saccharomyces cerevisiae, and the growth of the MpJNK is inhibited in the saccharomyces cerevisiae by using the RT-PCR technology, The specific results are as follows:1. Cloning and low-temperature expression of c-Jun N-terminal kinase (JNK) gene from the c-Jun N-terminal kinase (JNK) gene in the small-scale turtle shell. The c-Jun N-terminal kinase gene and MpJNK were obtained from the cDNA of Carapax Trionycis as a template. The bioinformatics analysis indicated that the total length of the JNK cDNA was 3413 bp, named MpJNK, which contained 1179 bp open reading frame,593 bp of 5 '-UTR and 1640 bp 3'-UTR, encoding 392 amino acids, belonging to the PKc-like superfamily, named MpJNK. The sequence has a conserved 13 amino acid sequence (IIHRDLKPSNIVV) at 145 to 157, a signature sequence for the activation site of the serine/ threonine protein kinase, and the predicted microcarapax JNK protein comprises an activation site, an ATP binding site and an activation ring (166-188), and contains a TPY (Thr-Pro-Tyr) phantom. The real-time fluorescence quantitative PCR results showed that, after 1 h at 4 鈩，
本文编号：2481940
[Abstract]:Microdera punctipennnis, a desert insect, is the endemic species of the quasi-step-in-one family of the Zhilerian basin of Xinjiang, and belongs to the non-freezing-type insects. The expression of c-Jun N-terminal kinase (JNK) in c-Jun N-terminal kinase (JNK) gene was detected in the low-temperature transcriptome database of the small chest and turtle shell, and the related gene of the kinase was obtained. It is known that the MAPK JNK signal transduction pathway is involved in the immune defense, but whether the pathway plays a role in low-temperature environmental stress is worthy of our in-depth study. The MpJNK gene and its related genes were obtained by using c-DNA as a template in the study. The anti-freezing function of the MpJNK protein is verified by using the RT-PCR technology, the anti-freezing function of the MpJNK protein is verified by using the Escherichia coli and the yeast expression system, the recombinant plasmid is transformed into the Saccharomyces cerevisiae, and the growth of the MpJNK is inhibited in the saccharomyces cerevisiae by using the RT-PCR technology, The specific results are as follows:1. Cloning and low-temperature expression of c-Jun N-terminal kinase (JNK) gene from the c-Jun N-terminal kinase (JNK) gene in the small-scale turtle shell. The c-Jun N-terminal kinase gene and MpJNK were obtained from the cDNA of Carapax Trionycis as a template. The bioinformatics analysis indicated that the total length of the JNK cDNA was 3413 bp, named MpJNK, which contained 1179 bp open reading frame,593 bp of 5 '-UTR and 1640 bp 3'-UTR, encoding 392 amino acids, belonging to the PKc-like superfamily, named MpJNK. The sequence has a conserved 13 amino acid sequence (IIHRDLKPSNIVV) at 145 to 157, a signature sequence for the activation site of the serine/ threonine protein kinase, and the predicted microcarapax JNK protein comprises an activation site, an ATP binding site and an activation ring (166-188), and contains a TPY (Thr-Pro-Tyr) phantom. The real-time fluorescence quantitative PCR results showed that, after 1 h at 4 鈩，
本文编号：2481940
本文链接：https://www.wllwen.com/kejilunwen/jiyingongcheng/2481940.html
最近更新
教材专著
