【摘要】：本文研究了西花薊馬的MAPK基因和蛋白表達(dá)純化,其適應(yīng)低溫脅迫或被真菌侵染的MAPK時(shí)空表達(dá)和磷酸化蛋白表達(dá)變化等分子生物學(xué)的內(nèi)容,為西花薊馬MAPK基因功能的發(fā)掘奠定基礎(chǔ),同時(shí)為闡明西花薊馬適應(yīng)低溫脅迫或被真菌侵染提供理論依據(jù)。主要研究結(jié)果如下:1.西花薊馬MAPK相關(guān)基因的分析與蛋白表達(dá)研究結(jié)果顯示,西花薊馬體內(nèi)JNKcDNA含有一個(gè)1191bpORF,編碼396個(gè)氨基酸,含有一個(gè)磷酸化激活基團(tuán)TPY的激活環(huán),在第18～314位氨基酸存在著一個(gè)典型的S_TKc結(jié)構(gòu)域,預(yù)測(cè)該成熟蛋白分子量為45.34kDa;p38cDNA含有一個(gè)840bpORF,編碼279個(gè)氨基酸,含有一個(gè)磷酸化激活基團(tuán)TGY的激活環(huán),在第20～270位氨基酸殘基區(qū)域存在著一個(gè)典型的S_TKc結(jié)構(gòu)域,預(yù)測(cè)該成熟蛋白分子量為31.64kDa;ERKcDNA含有一個(gè)987bpORF,編碼328個(gè)氨基酸,含有一個(gè)磷酸化激活基團(tuán)TEY的激活環(huán),在第2～276位氨基酸殘基區(qū)域存在著一個(gè)典型的S_TKc結(jié)構(gòu)域,預(yù)測(cè)該成熟蛋白分子量為38.34kDa;比對(duì)結(jié)果顯示磷酸化基團(tuán)TGY、TPY和TEY在不同物種中高度保守。并磷酸化序列高度相似,故可以使用商用抗體。2.低溫脅迫對(duì)西花薊馬MAPK信號(hào)通路的影響為探究低溫對(duì)西花薊馬MAPK信號(hào)通路的影響,設(shè)置了-13℃-39℃共5個(gè)溫度點(diǎn),利用Western blot技術(shù)進(jìn)行檢測(cè)。結(jié)果表明低溫脅迫激活西花薊馬體內(nèi)JNK、p38信號(hào)通路,并在0℃10min開(kāi)始磷酸化量明顯增多,而ERK無(wú)明顯變化。由此推測(cè),JNK和p38可能與西花薊馬低溫壓力下的應(yīng)激反應(yīng)有關(guān)。3.球孢白僵菌侵染對(duì)西花薊馬MAPK信號(hào)通路的影響采用實(shí)時(shí)熒光定量 PCR(quantitative real-time PCR,qRT-PCR)的2-△△Ct 相對(duì)定量法,對(duì)被球孢白僵菌感染的西花薊馬體內(nèi)JNK、p38和ERK基因的相對(duì)表達(dá)量進(jìn)行了測(cè)定。用p-JNK(磷酸化JNK)、p-p38(磷酸化p-p38)、和p-ERK(磷酸化ERK)商用抗體對(duì)以上處理樣品進(jìn)行檢測(cè)。與對(duì)照組相比,3個(gè)基因在西花薊馬被球孢白僵菌侵染后的相對(duì)表達(dá)量差異較大。JNK和p38在被球孢白僵菌侵染的西花薊馬若蟲(chóng)、蛹、成蟲(chóng)體內(nèi)的相對(duì)表達(dá)量均明顯上調(diào),其中在蛹期的相對(duì)表達(dá)量較高。JNK在蛹期36h相對(duì)表達(dá)量為對(duì)照組的7.25倍。p38在蛹期48h相對(duì)表達(dá)量為對(duì)照組的9.54倍。ERK在被球孢白僵菌侵染的西花薊馬若蟲(chóng)、蛹、成蟲(chóng)體內(nèi)的相對(duì)表達(dá)量顯示下調(diào)。利用Western blot技術(shù)對(duì)成蟲(chóng)被球孢白僵菌侵染后的12、24、36、48h時(shí)的樣品進(jìn)行檢測(cè)。結(jié)果表明被侵染后激活了西花薊馬體內(nèi)JNK、p38信號(hào)通路,可觀察到JNK和p38磷酸化蛋白在24h明顯增多并在48h時(shí)最大。而ERK磷酸化蛋白無(wú)明顯的變化。
[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，

本文編號(hào)：2508068