低温胁迫及球孢白僵菌侵染对西花蓟马体内JNK、p38和ERK蛋白磷酸化水平的影响
发布时间：2019-06-29 19:59
【摘要】：本文研究了西花蓟马的MAPK基因和蛋白表达纯化,其适应低温胁迫或被真菌侵染的MAPK时空表达和磷酸化蛋白表达变化等分子生物学的内容,为西花蓟马MAPK基因功能的发掘奠定基础,同时为阐明西花蓟马适应低温胁迫或被真菌侵染提供理论依据。主要研究结果如下:1.西花蓟马MAPK相关基因的分析与蛋白表达研究结果显示,西花蓟马体内JNKcDNA含有一个1191bpORF,编码396个氨基酸,含有一个磷酸化激活基团TPY的激活环,在第18～314位氨基酸存在着一个典型的S_TKc结构域,预测该成熟蛋白分子量为45.34kDa;p38cDNA含有一个840bpORF,编码279个氨基酸,含有一个磷酸化激活基团TGY的激活环,在第20～270位氨基酸残基区域存在着一个典型的S_TKc结构域,预测该成熟蛋白分子量为31.64kDa;ERKcDNA含有一个987bpORF,编码328个氨基酸,含有一个磷酸化激活基团TEY的激活环,在第2～276位氨基酸残基区域存在着一个典型的S_TKc结构域,预测该成熟蛋白分子量为38.34kDa;比对结果显示磷酸化基团TGY、TPY和TEY在不同物种中高度保守。并磷酸化序列高度相似,故可以使用商用抗体。2.低温胁迫对西花蓟马MAPK信号通路的影响为探究低温对西花蓟马MAPK信号通路的影响,设置了-13℃-39℃共5个温度点,利用Western blot技术进行检测。结果表明低温胁迫激活西花蓟马体内JNK、p38信号通路,并在0℃10min开始磷酸化量明显增多,而ERK无明显变化。由此推测,JNK和p38可能与西花蓟马低温压力下的应激反应有关。3.球孢白僵菌侵染对西花蓟马MAPK信号通路的影响采用实时荧光定量 PCR(quantitative real-time PCR,qRT-PCR)的2-△△Ct 相对定量法,对被球孢白僵菌感染的西花蓟马体内JNK、p38和ERK基因的相对表达量进行了测定。用p-JNK(磷酸化JNK)、p-p38(磷酸化p-p38)、和p-ERK(磷酸化ERK)商用抗体对以上处理样品进行检测。与对照组相比,3个基因在西花蓟马被球孢白僵菌侵染后的相对表达量差异较大。JNK和p38在被球孢白僵菌侵染的西花蓟马若虫、蛹、成虫体内的相对表达量均明显上调,其中在蛹期的相对表达量较高。JNK在蛹期36h相对表达量为对照组的7.25倍。p38在蛹期48h相对表达量为对照组的9.54倍。ERK在被球孢白僵菌侵染的西花蓟马若虫、蛹、成虫体内的相对表达量显示下调。利用Western blot技术对成虫被球孢白僵菌侵染后的12、24、36、48h时的样品进行检测。结果表明被侵染后激活了西花蓟马体内JNK、p38信号通路,可观察到JNK和p38磷酸化蛋白在24h明显增多并在48h时最大。而ERK磷酸化蛋白无明显的变化。
[Abstract]:In this paper, the expression and purification of MAPK gene and protein in thrips were studied, and the molecular biological contents such as temporal and spatial expression and phosphorylation protein expression of MAPK adapted to low temperature stress or fungal infection were studied, which laid a foundation for exploring the function of MAPK gene in thrips, and provided a theoretical basis for elucidating that thrips were adapted to low temperature stress or infected by fungi. The main results are as follows: 1. The analysis of MAPK related genes and protein expression in western flower thrips showed that JNKcDNA contained a 1191bpORF encoding 396amino acids and an activation ring of phosphorylation activating group TPY. There was a typical S_TKc domain at position 18 ~ 314. the molecular weight of the mature protein was predicted to be 45.34kDa. the results showed that there was a typical S_TKc domain at position 18 ~ 314. P38cDNA contains an 840bpORF encoding 279 amino acids and an activation ring of phosphorylation activating group TGY. There is a typical S_TKc domain in the region of amino acid residues at position 20 ~ 270. the molecular weight of the mature protein is predicted to be 31.64kV. ERKcDNA contains a 987bpORF encoding 328amino acids and an activation ring of phosphorylation activating group TEY. There is a typical S_TKc domain in the amino acid residue region of position 2 ~ 276. the molecular weight of the mature protein is predicted to be 38.34kDa. the comparison results show that the phosphorylation groups TGY,TPY and TEY are highly conserved in different species. The phosphorylation sequence is highly similar, so commercial antibodies can be used. 2. Effect of low temperature stress on MAPK signaling pathway of thrips in order to explore the effect of low temperature on MAPK signaling pathway of thrips, five temperature points at-13 鈩，
本文编号：2508068
[Abstract]:In this paper, the expression and purification of MAPK gene and protein in thrips were studied, and the molecular biological contents such as temporal and spatial expression and phosphorylation protein expression of MAPK adapted to low temperature stress or fungal infection were studied, which laid a foundation for exploring the function of MAPK gene in thrips, and provided a theoretical basis for elucidating that thrips were adapted to low temperature stress or infected by fungi. The main results are as follows: 1. The analysis of MAPK related genes and protein expression in western flower thrips showed that JNKcDNA contained a 1191bpORF encoding 396amino acids and an activation ring of phosphorylation activating group TPY. There was a typical S_TKc domain at position 18 ~ 314. the molecular weight of the mature protein was predicted to be 45.34kDa. the results showed that there was a typical S_TKc domain at position 18 ~ 314. P38cDNA contains an 840bpORF encoding 279 amino acids and an activation ring of phosphorylation activating group TGY. There is a typical S_TKc domain in the region of amino acid residues at position 20 ~ 270. the molecular weight of the mature protein is predicted to be 31.64kV. ERKcDNA contains a 987bpORF encoding 328amino acids and an activation ring of phosphorylation activating group TEY. There is a typical S_TKc domain in the amino acid residue region of position 2 ~ 276. the molecular weight of the mature protein is predicted to be 38.34kDa. the comparison results show that the phosphorylation groups TGY,TPY and TEY are highly conserved in different species. The phosphorylation sequence is highly similar, so commercial antibodies can be used. 2. Effect of low temperature stress on MAPK signaling pathway of thrips in order to explore the effect of low temperature on MAPK signaling pathway of thrips, five temperature points at-13 鈩，
本文编号：2508068
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