不同品系鸡akirin2和miR-155的克隆与表达特性分析

发布时间：2018-03-31 18:07

本文选题：鸡　切入点：akirin2基因　出处：《哈尔滨师范大学》2015年硕士论文

【摘要】：肌肉发育和抗病性状相关基因的筛选和鉴定,对于鸡的品种改良和育种具有重要的理论参考和利用价值。NF-κB是调控免疫应答和肌肉分化过程的重要转录因子,AKIRIN2通过间接作用NF-κB复合物,正向调控包括IL-6在内的多种基因的转录,并且akirin2基因与牛的肌内脂肪含量和禽类肌纤维的发育存在相关性;而miR-155是一个NF-κB下游调控的非编码小RNA,不但参与调控髓系细胞的增殖、活化和T、B细胞的分化,而且miR-155可以通过靶向肌肉发育相关基因而调控成肌细胞的分化。本研究选择与肌肉发育和免疫相关的靶基因akirin2和miR-155,分析其在不同品系鸡中的序列信息和表达分布特性,以期为鸡的品系改良和分子育种相关候选基因的筛选提供理论参考。主要研究方法和实验结果如下:新城疫弱毒苗免疫雏鸡的实验结果表明:免疫后7天时,akirin2在肝中高表达,其余组织表达变化不明显;免疫后14天时,akirin2在法氏囊中高表达,在肝中表达下调。研究结果暗示akirin2不仅参与了天然免疫反应,而且对雏鸡的适应性免疫也具有重要作用。不同品系鸡akirin2序列比较与表达特性分析结果表明:在四个品系鸡akirin2编码区的5'端存在4个SNP位点,这4个碱基突变导致相应位点三个氨基酸残基的改变。结构预测分析并未发现AKIRIN2蛋白二级结构的差异,但有几处蛋白结合位点的位置和数量发生了细微的改变,推测对AKIRIN2蛋白的表达和活性可能产生一定的影响。同一品系不同日龄鸡的表达分析结果显示,akirin2在各组织中广泛表达,在法氏囊、脾、血液等免疫组织中表达均为较高水平,随着日龄的增长,akirin2在肌肉中的表达呈现出了降低的趋势。不同品系同一日龄鸡的表达分析结果显示,akirin2在肌肉、心脏、肌胃和胸腺中的表达均较低,在血液、大肠、腺胃和肝脏中的表达均较高,但是在两个品系鸡的脂肪和脾等组织表现出了表达水平上的差异。miR-155的序列分析以及组织表达谱结果表明:三个品系鸡pri-miR-155的序列长度一致、序列无碱基缺失和突变。成熟miR-155在脊椎动物各纲中高度保守,同源性高达100%,而pre-mi R-155的同源性低于成熟miR-155。miR-155在三个品系鸡的各组织中广泛表达,无组织特异性,但在免疫相关组织中表达较高,而在肌肉组织中表达较低。
[Abstract]:Screening and identification of genes related to muscle development and disease resistance, NF- 魏 B is an important transcription factor for regulating immune response and muscle differentiation. AKIRIN2 regulates transcription of many genes, including IL-6, through indirect action of NF- 魏 B complex. Akirin2 gene is associated with the content of intramuscular fat and the development of poultry muscle fiber, and miR-155 is a non-coding small RNAs regulated downstream of NF- 魏 B, which not only participates in the regulation of proliferation, activation and differentiation of myeloid cells. Moreover, miR-155 can regulate the differentiation of myoblasts by targeting the genes associated with muscle development. In this study, the target genes akirin2 and miR-155, which are related to muscle development and immunity, were selected to analyze the sequence information and expression distribution of these genes in different strains of chicken. The main research methods and experimental results are as follows: the experimental results of Newcastle disease attenuated vaccine immunized chicks showed that: 7 days after immunization, the expression of Aakirin2 in the liver was high. There was no significant change in the expression of akirin2 in the other tissues, and the expression of kirin2 was highly expressed in bursa of Fabricius and down-regulated in liver 14 days after immunization. The results suggested that akirin2 was not only involved in innate immune response, but also in the liver. The results of akirin2 sequence comparison and expression analysis showed that there were four SNP loci at the 5 'end of akirin2 coding region in four strains of chickens. These four base mutations led to the change of three amino acid residues at the corresponding site. No difference in secondary structure of AKIRIN2 protein was found in the structural prediction analysis, but there were slight changes in the position and number of several protein binding sites. The expression and activity of AKIRIN2 protein may be affected by the same strain of different day-old chickens. The results showed that the expression of AKIRIN2 2 was widely expressed in all tissues, and the expression level was higher in immune tissues such as bursa of Fabricius, spleen, blood and so on. With the age of day, the expression of anakirin2 in muscle showed a decreasing trend. The results of expression analysis of different strains of chicken of the same day showed that the expression of pakirin2 was lower in muscle, heart, muscle stomach and thymus, and was found in blood and large intestine. The expression of adenogastric and liver was higher, but the sequence analysis and the tissue expression profile of the difference in the expression level of fat and spleen showed that the sequence length of pri-miR-155 was the same among the three strains of chicken. The homology of pre-mi R-155 was lower than that of mature miR-155.miR-155 in all tissues of three strains of chicken. However, the expression was higher in immune-related tissues and lower in muscle tissues.
【学位授予单位】：哈尔滨师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831

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