黄翅大白蚁几丁质酶基因鉴定及功能性表达

发布时间：2018-03-22 05:18

本文选题：黄翅大白蚁　切入点：几丁质酶基因　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：白蚁是一种社会性昆虫。因为它们对于木质纤维素强大的降解能力,所以在生态圈的碳循环中发挥重要的作用。根据肠道共生物中是否包含原生动物,可将白蚁分成低等白蚁和高等白蚁。高等白蚁中与真菌共生的白蚁为培菌白蚁。培菌白蚁的食物中包含真菌孢子,而孢子的细胞壁主要成分是几丁质,故培菌白蚁肠道中可能存在几丁质酶来起着降解真菌孢子细胞壁的作用。黄翅大白蚁是培菌白蚁的一种,目前对培菌白蚁来源昆虫几丁质酶的研究是非常有限的。分析黄翅大白蚁肠道转录组测序结果后,我们从中筛选出功能注释为几丁质酶的基因,对它们进行生物信息学分析。本研究主要对其中可能参与代谢的几丁质酶基因进行了基因克隆和功能性表达,具体内容如下:1.本文先对黄翅大白蚁转录组测序结果进行分析,通过关键词"chitinase"搜索,发现有40个功能注释为几丁质酶的unigenes。将这40个几丁质酶基因进行一系列生物信息学分析,并对其中三个基因进行进一步功能的探索。2.Unigene基因comp133624_c0的在前肠中相对表达量最高,为277.21。该片段进行生物信息学分析之后,设计引物,通过PCR得到cDNA全长,在大肠杆菌JM109中表达;unigene基因comp174658_c0的中肠相对表达量最高,523.34。对该片段进行生物信息学分析之后,设计引物,通过PCR得到cDNA全长,然后将该基因在大肠杆菌JM109中和BL21(DE3)以及毕赤酵母中异源表达;unigene基因comp182537-_c0在中肠和后肠的相对表达量都相对较高,分别为750.43和1638.2。该片段编码一个含有CBM-14(结合域)的几丁质酶,并且设计引物,通过PCR得到cDNA全长并克隆到在大肠杆菌JM109中和BL21(DE3)表达。3.为了进步一确认上述三个基因的优势表达部位,我们还对上述基因进行了qPCR和RT-PCR验证,证实是否与转录组测序结果的优势表达位点相吻合。总之,本研究成功克隆得到三个几丁质酶基因,而且在大肠杆菌异源表达。它们的重组蛋白均能检测到几丁质酶活性,但是几丁质酶活性不高。这种现象可能是因为昆虫来源蛋白在大肠杆菌中表达时,重组蛋白没得到很好的修饰和折叠,导致大多数重组蛋白都形成了包涵体。另外,unigene基因comp174658_c0在毕赤酵母中异源表达且表达产物具有降解几丁质的能力。最后,qPCR和RT-PCR的结果显示:除了基因comp174658_c0外,另外两个基因在各个部位的表达水平趋势与转录组测序结果相符合。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q966

【相似文献】