东北虎肝cDNA文库的构建、EST表达序列析及相关基因功能分析

发布时间：2018-05-27 04:22

本文选题：东北虎 + 肝脏　；参考：《东北林业大学》2016年硕士论文

【摘要】：东北虎(Panthera tigris altaica)是国家Ⅰ级重点保护动物,也是全球最濒危的物种之一。长期以来,东北虎生存环境的严重破坏,猎物资源匮乏,人类乱捕乱杀因素,导致野生东北虎处于绝灭的境地,及时开展东北虎遗传资源保存及相关分子生物学研究,具有非常重要科学价值和保护意义。本文选取圈养东北虎的肝脏为研究材料,采用现代分子生物学的实验方法,构建了东北虎肝脏的cDNA文库,同时对该cDNA文库的质量加以分析鉴定,获得出如下的研究结果：1、以2.5岁龄的东北虎胎肝脏组织为研究材料,获得了高质量东北虎胎盘cDNA文库。原始文库库容量为2.68×106pfu/ml,重组率为97.2%,扩增后的文库库容量达到4.8×109pfu/ml,重组率为94.6%,随机挑选200个阳性菌斑进行PCR检测,获得的文库中插入片段的长度范围在0.5～3.0Kb之间,并且片段的平均长度为0.78Kb。2、通过随机挑选200个克隆展开测序工作,运用GENE BANK中的BLAST对测得的序列进行比对分析,获得了138个原始序列,去除掉测序质量比较差和很短(200bp)的序列,最后获得了105条有效的ESTs序列。大部分序列长度都大于300bp,主要集中的在600～900bp之间,测序成功率达到89.3%。这些序列经过GO分析分成三大类,即细胞组成,分子功能,生物进程。3、在GO分类结果中,80个序列中,有71个被注释,10种分子功能被记录了107次,有12个参与的生物学过程以及6种细胞组分。GO分类发现同基因有不同的分子功能,也能参与不同的生物学过程,这反映了东北虎生命过程的复杂性。4、将80个基因进行蛋白质分类及代谢通路分析,得到24种蛋白质及33个信号通路。并对重要的蛋白质及代谢通路进行分析。5、通过构建肝组织相关基因相互作用的网络图,并运用GO注释,KEGG代谢途径分析和InterPro检索等工具以及对相关文献的查找,筛选出肝组织中相关的基因,例如SOD1,CTNNB1,LIF2等,对这些基因的功能进行研究,从而能够提高对于东北虎新陈代谢的生物学角度的认知程度,增强东北虎自身体质并且提高其生存质量。
[Abstract]:Panthera tigris altaica) is one of the most endangered species in the world. For a long time, the serious destruction of the living environment of the Siberian tiger, the scarcity of prey resources, and the human random hunting and killing factors have led to the extinction of the wild Siberian tiger. The preservation of the genetic resources of the Siberian tiger and the related molecular biology research have been carried out in time. It has very important scientific value and protective significance. In this paper, the liver of the captive Siberian tiger was selected as the research material, and the cDNA library of the liver of the Siberian tiger was constructed by using the modern molecular biology method, and the quality of the cDNA library was analyzed and identified. The following results were obtained: 1: 1. A high quality placental cDNA library of Amur tiger was obtained from the liver tissue of a 2.5 year old Siberian tiger fetus. The original library capacity was 2.68 脳 106pfuP / ml, the recombination rate was 97.2ml, the expanded library capacity reached 4.8 脳 109pfuP / ml, and the recombination rate was 94.6. 200 positive plaque were randomly selected for PCR detection, and the length of inserted fragments in the library was between 0.5~3.0Kb. And the average length of the fragment is 0.78 Kb. 2. By randomly selecting 200 clones to carry out the sequencing work, using BLAST in GENE BANK to analyze the measured sequence, 138 original sequences were obtained, and the sequence of poor quality and very short 200bp was removed. Finally, 105 valid ESTs sequences are obtained. Most of the sequences were longer than 300 BP, mainly between 600~900bp and the success rate of sequencing was 89.33%. These sequences are classified into three categories by go analysis: cell composition, molecular function, biological process. Out of 80 sequences, 71 have been annotated for 10 molecular functions 107 times. There are 12 involved biological processes and 6 cell fractions. Go classification shows that the same gene has different molecular functions and can also participate in different biological processes. This reflects the complexity of the life process of Amur tiger. The protein classification and metabolic pathway analysis of 80 genes obtained 24 proteins and 33 signal pathways. The important proteins and metabolic pathways were analyzed. 5. By constructing a network map of the interaction of genes related to liver tissue, and using go annotated KEGG metabolic pathway analysis and InterPro search tools, and searching the relevant literature, and so on. The related genes in liver tissue, such as SOD1 CTNNB1LIF2, were screened, and the function of these genes was studied, so as to improve the cognition of the biological angle of Amur tiger metabolism, enhance the physical constitution and improve the quality of life of Amur tiger.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q953

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