【摘要】：本文研究了西花薊馬的MAPK基因和蛋白表達純化,其適應(yīng)低溫脅迫或被真菌侵染的MAPK時空表達和磷酸化蛋白表達變化等分子生物學的內(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研究結(jié)果顯示,西花薊馬體內(nèi)JNKcDNA含有一個1191bpORF,編碼396個氨基酸,含有一個磷酸化激活基團TPY的激活環(huán),在第18～314位氨基酸存在著一個典型的S_TKc結(jié)構(gòu)域,預(yù)測該成熟蛋白分子量為45.34kDa;p38cDNA含有一個840bpORF,編碼279個氨基酸,含有一個磷酸化激活基團TGY的激活環(huán),在第20～270位氨基酸殘基區(qū)域存在著一個典型的S_TKc結(jié)構(gòu)域,預(yù)測該成熟蛋白分子量為31.64kDa;ERKcDNA含有一個987bpORF,編碼328個氨基酸,含有一個磷酸化激活基團TEY的激活環(huán),在第2～276位氨基酸殘基區(qū)域存在著一個典型的S_TKc結(jié)構(gòu)域,預(yù)測該成熟蛋白分子量為38.34kDa;比對結(jié)果顯示磷酸化基團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.低溫脅迫對西花薊馬MAPK信號通路的影響為探究低溫對西花薊馬MAPK信號通路的影響,設(shè)置了-13℃-39℃共5個溫度點,利用Western blot技術(shù)進行檢測。結(jié)果表明低溫脅迫激活西花薊馬體內(nèi)JNK、p38信號通路,并在0℃10min開始磷酸化量明顯增多,而ERK無明顯變化。由此推測,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.球孢白僵菌侵染對西花薊馬MAPK信號通路的影響采用實時熒光定量 PCR(quantitative real-time PCR,qRT-PCR)的2-△△Ct 相對定量法,對被球孢白僵菌感染的西花薊馬體內(nèi)JNK、p38和ERK基因的相對表達量進行了測定。用p-JNK(磷酸化JNK)、p-p38(磷酸化p-p38)、和p-ERK(磷酸化ERK)商用抗體對以上處理樣品進行檢測。與對照組相比,3個基因在西花薊馬被球孢白僵菌侵染后的相對表達量差異較大。JNK和p38在被球孢白僵菌侵染的西花薊馬若蟲、蛹、成蟲體內(nèi)的相對表達量均明顯上調(diào),其中在蛹期的相對表達量較高。JNK在蛹期36h相對表達量為對照組的7.25倍。p38在蛹期48h相對表達量為對照組的9.54倍。ERK在被球孢白僵菌侵染的西花薊馬若蟲、蛹、成蟲體內(nèi)的相對表達量顯示下調(diào)。利用Western blot技術(shù)對成蟲被球孢白僵菌侵染后的12、24、36、48h時的樣品進行檢測。結(jié)果表明被侵染后激活了西花薊馬體內(nèi)JNK、p38信號通路,可觀察到JNK和p38磷酸化蛋白在24h明顯增多并在48h時最大。而ERK磷酸化蛋白無明顯的變化。
[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：2508068