本文選題：黃翅大白蟻　切入點(diǎn)：幾丁質(zhì)酶基因　出處：《山東大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：白蟻是一種社會(huì)性昆蟲(chóng)。因?yàn)樗鼈儗?duì)于木質(zhì)纖維素強(qiáng)大的降解能力,所以在生態(tài)圈的碳循環(huán)中發(fā)揮重要的作用。根據(jù)腸道共生物中是否包含原生動(dòng)物,可將白蟻分成低等白蟻和高等白蟻。高等白蟻中與真菌共生的白蟻為培菌白蟻。培菌白蟻的食物中包含真菌孢子,而孢子的細(xì)胞壁主要成分是幾丁質(zhì),故培菌白蟻腸道中可能存在幾丁質(zhì)酶來(lái)起著降解真菌孢子細(xì)胞壁的作用。黃翅大白蟻是培菌白蟻的一種,目前對(duì)培菌白蟻來(lái)源昆蟲(chóng)幾丁質(zhì)酶的研究是非常有限的。分析黃翅大白蟻腸道轉(zhuǎn)錄組測(cè)序結(jié)果后,我們從中篩選出功能注釋為幾丁質(zhì)酶的基因,對(duì)它們進(jìn)行生物信息學(xué)分析。本研究主要對(duì)其中可能參與代謝的幾丁質(zhì)酶基因進(jìn)行了基因克隆和功能性表達(dá),具體內(nèi)容如下:1.本文先對(duì)黃翅大白蟻轉(zhuǎn)錄組測(cè)序結(jié)果進(jìn)行分析,通過(guò)關(guān)鍵詞"chitinase"搜索,發(fā)現(xiàn)有40個(gè)功能注釋為幾丁質(zhì)酶的unigenes。將這40個(gè)幾丁質(zhì)酶基因進(jìn)行一系列生物信息學(xué)分析,并對(duì)其中三個(gè)基因進(jìn)行進(jìn)一步功能的探索。2.Unigene基因comp133624_c0的在前腸中相對(duì)表達(dá)量最高,為277.21。該片段進(jìn)行生物信息學(xué)分析之后,設(shè)計(jì)引物,通過(guò)PCR得到cDNA全長(zhǎng),在大腸桿菌JM109中表達(dá);unigene基因comp174658_c0的中腸相對(duì)表達(dá)量最高,523.34。對(duì)該片段進(jìn)行生物信息學(xué)分析之后,設(shè)計(jì)引物,通過(guò)PCR得到cDNA全長(zhǎng),然后將該基因在大腸桿菌JM109中和BL21(DE3)以及畢赤酵母中異源表達(dá);unigene基因comp182537-_c0在中腸和后腸的相對(duì)表達(dá)量都相對(duì)較高,分別為750.43和1638.2。該片段編碼一個(gè)含有CBM-14(結(jié)合域)的幾丁質(zhì)酶,并且設(shè)計(jì)引物,通過(guò)PCR得到cDNA全長(zhǎng)并克隆到在大腸桿菌JM109中和BL21(DE3)表達(dá)。3.為了進(jìn)步一確認(rèn)上述三個(gè)基因的優(yōu)勢(shì)表達(dá)部位,我們還對(duì)上述基因進(jìn)行了qPCR和RT-PCR驗(yàn)證,證實(shí)是否與轉(zhuǎn)錄組測(cè)序結(jié)果的優(yōu)勢(shì)表達(dá)位點(diǎn)相吻合。總之,本研究成功克隆得到三個(gè)幾丁質(zhì)酶基因,而且在大腸桿菌異源表達(dá)。它們的重組蛋白均能檢測(cè)到幾丁質(zhì)酶活性,但是幾丁質(zhì)酶活性不高。這種現(xiàn)象可能是因?yàn)槔ハx(chóng)來(lái)源蛋白在大腸桿菌中表達(dá)時(shí),重組蛋白沒(méi)得到很好的修飾和折疊,導(dǎo)致大多數(shù)重組蛋白都形成了包涵體。另外,unigene基因comp174658_c0在畢赤酵母中異源表達(dá)且表達(dá)產(chǎn)物具有降解幾丁質(zhì)的能力。最后,qPCR和RT-PCR的結(jié)果顯示:除了基因comp174658_c0外,另外兩個(gè)基因在各個(gè)部位的表達(dá)水平趨勢(shì)與轉(zhuǎn)錄組測(cè)序結(jié)果相符合。
[Abstract]:Termites are social insects. Because of their ability to degrade lignocellulose, termites play an important role in the carbon cycle of the biosphere. The termites can be divided into lower termites and higher termites. The higher termites that are symbiotic with fungi are cultured termites. The food of cultured termites contains fungal spores, and the cell walls of spores are mainly chitin. Therefore, chitinase may exist in the intestinal tract of cultured termites to degrade the cell wall of fungal spores. The study of chitinase from termites is very limited. After analyzing the results of transcriptome sequencing of termites yellowfin, we have screened out the genes whose function is annotated as chitinase. In this study, the chitinase genes that may be involved in metabolism were cloned and functionally expressed, and the specific contents were as follows: 1. The results of transcriptome sequencing of termites yellowfin were analyzed in this paper. By searching for the key word "chitinase", we found that there were 40 unigeneses whose function was annotated as chitinase. The 40 chitinase genes were analyzed by a series of bioinformatics. The relative expression of Unigene gene comp133624_c0 in the foregut was 277.21. After bioinformatics analysis, a primer was designed to obtain the full length of cDNA by PCR. In Escherichia coli (E. coli) JM109, the relative expression level of comp174658_c0 was the highest in the midgut. After bioinformatics analysis of the fragment, primers were designed to obtain the full length of cDNA by PCR. Then the relative expression of the gene in Escherichia coli JM109 and BL21DDE3) and in Pichia pastoris was higher than that in midgut and hindgut, 750.43 and 1638.2, respectively. The fragment encodes a chitinase containing CBM-14 (binding domain). The full length of cDNA was obtained by PCR and cloned into E. coli JM109 and BL21DE3) expression. In conclusion, three chitinase genes were successfully cloned and expressed in Escherichia coli. All of their recombinant proteins could detect chitinase activity. But chitinase activity is low. This phenomenon may be due to the fact that when insect derived proteins are expressed in E. coli, the recombinant proteins are not well modified and folded. Most of the recombinant proteins form inclusion bodies. In addition, the gene comp174658_c0 is heterologous expressed in Pichia pastoris and the expressed product has the ability to degrade chitin. Finally, the results of QPCR and RT-PCR show that in addition to the gene comp174658_c0, The expression level of the other two genes was consistent with the results of transcriptome sequencing.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q78;Q966

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