發(fā)布時(shí)間：2019-05-21 07:39

【摘要】：荒漠昆蟲小胸鱉甲(Microdera punctipennisnnis)作為新疆準(zhǔn)噶爾盆地的擬步甲科的特有種,屬避凍型昆蟲。在小胸鱉甲低溫轉(zhuǎn)錄組數(shù)據(jù)庫(kù)中分析發(fā)現(xiàn)c-Jun N端激酶(c-Jun N-terminal kinase,JNK)基因上調(diào)表達(dá),并獲得該激酶的相關(guān)基因。已知該MAPK JNK信號(hào)轉(zhuǎn)導(dǎo)通路參與免疫防御作用,然而該通路在低溫環(huán)境脅迫時(shí)是否發(fā)揮作用,值得我們深入研究。本研究以小胸鱉甲的c DNA作為模板,克隆得到MpJNK基因及其相關(guān)基因。利用RT-PCR技術(shù)分別分析不同時(shí)間不同溫度的表達(dá)譜,利用大腸桿菌和酵母表達(dá)系統(tǒng),驗(yàn)證MpJNK蛋白的抗凍功能,通過(guò)將重組質(zhì)粒轉(zhuǎn)化至釀酒酵母,利用RT-PCR技術(shù)檢測(cè)出MpJNK在釀酒酵母中抑制了其生長(zhǎng),具體結(jié)果如下:1.小胸鱉甲c-Jun N端激酶(c-Jun N-terminal kinase,JNK)基因的克隆與低溫表達(dá)譜。以小胸鱉甲cDNA為模板克隆獲得c-Jun N端激酶基因,MpJNK。生物信息學(xué)分析表明,小胸鱉甲JNK cDNA全長(zhǎng)3413bp,命名為MpJNK,它包含1179bp的開(kāi)放閱讀框、593bp的5’-UTR和1640bp的3’-UTR,編碼392個(gè)氨基酸,屬于PKc like超家族,命名為MpJNK。MpJNK與赤擬谷盜JNK的同源性達(dá)97%。該序列在145~157處有保守的13個(gè)氨基酸序列(IIHRDLKPSNIVV),為絲氨酸/蘇氨酸蛋白激酶激活位點(diǎn)的簽名序列,預(yù)測(cè)小胸鱉甲JNK蛋白包含一個(gè)激活位點(diǎn),ATP結(jié)合位點(diǎn)以及活化環(huán)(166-188),并含有一個(gè)TPY(Thr-Pro-Tyr)的模體。實(shí)時(shí)熒光定量PCR結(jié)果顯示,在4℃和-4℃低溫脅迫1h后,MpJNK的mRNA水平都顯著高于室溫對(duì)照,并呈現(xiàn)先升高后降低的應(yīng)激響應(yīng)趨勢(shì),該基因是冷響應(yīng)基因,對(duì)小胸鱉甲應(yīng)對(duì)低溫脅迫可能發(fā)揮作用。2.JNK信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)基因的低溫表達(dá)譜分析。通過(guò)對(duì)小胸鱉甲JNK信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)基因在不同時(shí)間低溫脅迫下的相對(duì)表達(dá)量變化進(jìn)行檢測(cè),實(shí)時(shí)熒光定量PCR結(jié)果顯示,4℃以及-4℃脅迫小胸鱉甲成蟲0.3~11 h后,MpJNK相關(guān)基因的mRNA水平的變化呈現(xiàn)出位于細(xì)胞膜上的JNK信號(hào)轉(zhuǎn)導(dǎo)通路的相關(guān)基因均發(fā)生不同程度的上調(diào)表達(dá),且相對(duì)表達(dá)量的倍數(shù)高于MAPKKK(包括MLK、TAK、POSH、MEKK),MAPKK(包括MKK4、LZK)以及MAPK(包括JNK、STAT、smad和nfat),即小胸鱉甲jnk信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)基因?qū)Φ蜏乩漤憫?yīng)的表達(dá)趨勢(shì)可概括為從細(xì)胞膜到細(xì)胞質(zhì)最終到達(dá)細(xì)胞核的mrna的相對(duì)表達(dá)量伴隨jnk信號(hào)轉(zhuǎn)導(dǎo)通路的級(jí)聯(lián)模式呈逐級(jí)遞減趨勢(shì)。3.mpjnk的原核表達(dá)及其增強(qiáng)細(xì)菌抗凍功能的驗(yàn)證。將mpjnk構(gòu)建于pet28a載體上,轉(zhuǎn)化大腸桿菌bl21(de3),然后篩選陽(yáng)性單克隆bl21(pet28a-mpjnk),誘導(dǎo)表達(dá)獲得可溶性蛋白。利用westernblot印跡技術(shù)結(jié)果顯示,重組蛋白his-mpjnk表達(dá)正確。實(shí)驗(yàn)菌與對(duì)菌在-18℃的生長(zhǎng)曲線結(jié)果顯示,重組的實(shí)驗(yàn)菌生長(zhǎng)優(yōu)于對(duì)照菌。表明his-mpjnk可能賦予大腸桿菌bl21(pet28a-mpjnk)的抗凍能力。4.采用真核表達(dá)系統(tǒng)進(jìn)行mpjnk蛋白的功能研究。將mpjnk構(gòu)建至pyes2載體上,轉(zhuǎn)化至釀酒酵母(invscⅠ)中,并獲得實(shí)驗(yàn)菌invscⅠ(pyes2-mpjnk),對(duì)照組為載體pyes2轉(zhuǎn)化菌invscⅠ(pyes2)中,半乳糖用以誘導(dǎo)外源蛋白mpjnk表達(dá)。-18℃不同時(shí)間低溫脅迫實(shí)驗(yàn)菌、對(duì)照菌生長(zhǎng)曲線結(jié)果表明,對(duì)照菌的生長(zhǎng)優(yōu)于實(shí)驗(yàn)菌;僅僅脅迫5d后試驗(yàn)菌便表現(xiàn)出停止生長(zhǎng),該結(jié)果表明酵母表達(dá)的mpjnk可能抑制了脅迫的耐受性。檢測(cè)實(shí)驗(yàn)菌invscⅠ(pyes2-mpjnk)和對(duì)照組菌invscⅠ(pyes2)低溫脅迫下的抗逆性指標(biāo)脯氨酸,可溶性糖及甘油含量,實(shí)驗(yàn)結(jié)果顯示對(duì)照菌的抗逆性指標(biāo)高于實(shí)驗(yàn)組。表明外源基因的插入可能抑制了酵母抗逆性生理指標(biāo)的合成。利用核酸免疫小鼠制備mpjnk蛋白的特異性抗體,獲得mpjnk小鼠抗血清,效價(jià)為1:12800,以此為一抗進(jìn)行westernblot,檢測(cè)invscⅠ(pyes2-mpjnk)超聲后的裂解液,酵母外源蛋白在目的條帶處正確表達(dá)。5.酵母系統(tǒng)中與jnk信號(hào)通路同源的hog信號(hào)通路相關(guān)基因的低溫表達(dá)譜。利用實(shí)時(shí)熒光定量pcr對(duì)實(shí)驗(yàn)菌和對(duì)照菌hog信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)基因在不同時(shí)間低溫脅迫下的相對(duì)表達(dá)量變化進(jìn)行檢測(cè),結(jié)果顯示4℃和-18℃脅迫重組酵母菌5min至8d,hog相關(guān)基因的mrna水平的變化在短時(shí)間內(nèi)(5min~60min)呈現(xiàn)出不同程度的上調(diào)表達(dá),然而和對(duì)照菌相比較在1~8d時(shí)呈現(xiàn)出顯著性下調(diào),表明在重組釀酒酵母中,hog信號(hào)通路的相關(guān)基因的表達(dá)被抑制。通過(guò)研究我們發(fā)現(xiàn),JNK(c-Jun氨基末端激酶)作為MAPK(絲裂原活化蛋白激酶)家族的一員,不僅能夠通過(guò)轉(zhuǎn)錄和非轉(zhuǎn)錄的依賴形式,活化下游核轉(zhuǎn)錄因子,介導(dǎo)器官發(fā)育期的細(xì)胞改變,而且將其構(gòu)建于不同的載體轉(zhuǎn)化大腸桿菌以及釀酒酵母后也能響應(yīng)低溫的脅迫。
[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，

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