牛T1R1基因的克隆及其组织差异表达分析
时间:2019-08-17 11:44 来源:毕业论文 作者:毕业论文 点击:次
| 摘要：许多动物实验都验证了T1R1 这一基因在舌头、肌肉中表达量很高，而且在其他的组织里也有一定的含量，同氨基酸的代谢调控有关。为此，本文用PCR扩增的方法得到T1R1基因的目的片段并通过测序验证结果是否可靠。并且以实时荧光定量 PCR 的方法检测了在奶牛乳腺、心、肝、脾、肺、肾、舌头、背最长肌、腿肌中 T1R1 基因的表达情况及其规律。 38505 通过多项重复实验，我得到了如下结果： 1.证实T1R1在奶牛组织中表达。 经BLAST同源性比对后发现，克隆片段与反刍动物的T1R1基因的同源性分别为野牛（99%）、普通牛（99%）、牦牛（99%）；和人及其他单胃动物相比，同源性在80%—83%之间，基本可以确认克隆出的是奶牛的T1R1基因，可在奶牛各组织中表达。 2.奶牛的各组织中 T1R1 基因的表达具有显著差异。 奶牛各组织中的T1R1基因的表达具有明显的不同含量。其与乳腺组织比较后得出的表达量（2-△△CT值）从高到低分别为腿肌（5.38）、背最长肌（3.61）、心脏（2.07）、舌头（1.59）、乳腺（1.00）、肺（0.33）、肝脏（0.06）、肾脏（0.05）、脾脏（0.04），奶牛的各组织之间表达差异十分显著(P<0.05)。 毕业论文关键词：奶牛； T1R1基因； 荧光定量PCR； 氨基酸受体 Cattle T1R1 gene cloning and analysis of differentially expressed in the organization Abstract T1R1 genes play an important role for identifying the amino acid and its ligand is amino acids. More and more experiments suggest that the T1R1 genes besides expressed in taste buds, also expressed in other organizations, and in the form of heterologous dimers play a role, in the absorption of amino acids adjust. Therefore, this article use the method of PCR amplification of T1R1 gene fragments and validated by sequencing results are reliable. And in real time fluorescence quantitative PCR method to detect the cow mammary gland, heart, liver, spleen, lung, kidney, tongue, Longissimus Dorsi, leg muscle in T1R1 gene expression and regulation. Through the experiment I got the following results: 1. The target fragment can be expressed in many dairy cow organs. After BLAST homology ratio found in cow T1R1 gene as a reference, in ruminant animals T1R1 gene homology of bison respectively (99%), ordinary cow (99%), Bos mutus (99%);Compared to people and other single stomach animals, homology between 80% - 83%, we can confirm T1R1 gene is cloned that can be expressed in dairy cows each organization. 2. The cow T1R1 gene expression differed between organs. mRNA expression of cow T1R1 gene in each group showed obvious difference. The relative expression quantity (2 - delta delta CT value) from high to low in order for the leg muscles (5.38), the longest back muscle (3.61), heart (2.07), tongue (1.59), mammary gland (1.00), lung (0.33), liver (0.06), (0.05) of kidney, spleen (0.04), organizations expressed significant difference (P < 0.05). Key Words: dairy cattle; T1R1 gene; amino acid receptor; real time-PCR. 目录 引言 5 1材料与方法 6 1.1实验的材料 6 1.1.1试剂 6 1.1.4实验的仪器 6 1.2实验的方法 7 1.2.1采集实验样本 7 1.2.2 提取各组织的总RNA 7 1.2.3.1 反转录反应 8 1.2.4.PCR扩增 8 1.2.5 PCR反应终产物的凝胶电泳检测 9 1.2.6 目的基因片段的扩增和回收 10 1.2.7. 胶回收与连接载体 10 1.2.8 阳性重组子的鉴定 11 1.2.9.质粒的提取 12 2 结果与数据分析 12 2.1.提取的总RNA 12 (责任编辑：qin) |