家犬苦味受体基因族的进化和功能研究
文献类型：学位论文
| 作者 | 胡玲玲 |
| 学位类别 | 博士 |
| 答辩日期 | 2013-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 施鹏 |
| 关键词 | 苦味受体基因 家犬 进化 基因功能 配体 |
| 其他题名 | Evolutionary and Functional Study of Bitter Taste Receptor (T2R) Gene Family of Dogs |
| 学位专业 | 遗传学 |
| 中文摘要 | 苦味识别能力主要检测苦味有毒物质，是一种避免生物体受到苦味有毒物质毒害的防御机制。由苦味受体基因编码的苦味受体被苦味物质激活后，通过下游信号通路将外界的苦味信号传导到大脑皮层，引起厌恶反应，避免动物摄入有毒物质。食肉类动物食物主要来源于动物组织，含有较少的苦味有毒物质，苦味识别能力不太重要。家犬，作为第一个被人工驯化的家养动物，食物中含有较少苦味有毒物质，苦味识别能力在它们的生活中不再重要。因此我们提出以下假说：家犬苦味受体基因受到放松的选择压力。我们通过研究苦味受体基因家族在家犬群体中的微进化模式和苦味受体的功能来验证该假说。本研究首先扩增来自15个家犬品系的30只家犬的苦味受体基因家族15个成员、5个苦味受体假基因和16个随机选取的基因间序列，然后比较家犬和其它胎盘哺乳类苦味受体基因家族微进化的异同并研究家犬苦味受体基因家族的功能和相关的功能位点。通过对家犬的苦味受体基因家族的群体遗传学研究，首次揭示家犬的苦味受体基因家族整体的群体遗传多样性很低，低于基因间序列的遗传多样性。为了进一步探讨家犬不同类型苦味受体基因的微进化模式，我们把家犬的苦味受体基因分为三类，发现家犬的多对多直系同源基因的遗传多样性最高而一对一直系同源基因遗传多样性最低，说明不同苦味受体基因受到不同的选择压力。这个结论同样适用于另外三类胎盘哺乳类的苦味受体。我们发现dT2R14的群体遗传多样性高于基因间序列的遗传多样性，在研究的30个样本中存在17个单倍型，可能受到放松的纯净化或多样化选择。为了揭示家犬整个基因家族的功能，我们利用体外细胞异源表达功能实验平台，异源表达家犬苦味受体基因家族的所有成员。我们用33种苦味物质对所有家犬苦味受体进行功能筛选试验。我们首次报道家犬的3个苦味受体的5个配体。这3个苦味受体分别是dTas2R1、dTas2R10和dTas2R14，其中dTas2R1的三个配体分别是秋水仙素(Colchicine)、(-)-α-Thujone和3-Methyl-1-pentyn-3-ol；dTas2R10的配体是Diethylene triamine pentaacetic acid (DTPA)；dTas2R14的配体是6-Propyl-2-thiouracil (PROP)。通过比较和PROP结构类似的化学物质激活dTas2R14受体的程度，我们发现PROP结构中激活dTas2R14受体的重要结构。由于dT2R14在群体中的有17个等位基因并且在人中的同源基因是hT2R43-44基因簇，该基因簇的配体已被报道。通过研究该基因不同等位基因功能来了解家犬苦味受体基因在群体中功能的分化。我们异源表达dTas2R14的17个单倍型，通过功能实验发现dTas2R14的第二个天然的配体amarogentin，并且发现dTas2R14对PROP和amarogentin的反应一致。该基因的17个单倍型分成两大类，一类是有功能的13个单倍型，能被这两个配体激活；另一类没有功能的4个单倍型，不能被这两种配体激活。有功能的单倍型的第91位氨基酸都是丝氨酸，没有功能的单倍型在91位氨基酸都是天冬酰胺。通过对91位氨基酸构建点突变的dTas2R14的单倍型的功能实验发现，91位氨基酸是dTas2R14发挥功能的关键位点。同时发现另外两个氨基酸位点175和185也能影响dTas2R14的功能。本研究为理解苦味受体基因的多态性和受体蛋白结构功能多样性提供了新的思路。 |
| 英文摘要 | Bitter taste perception detects bitter toxic sunstances and prevents organisms against bitter toxins which is a defense mechanism. The comnbination of bitter taste receptor coded by bitter taste receptor genes (T2Rs) with bitter ligands trigger the downstream signaling pathway and transduct external signal to cerebral cortex which cause aversion behavior to avoiding having toxic substances. Carnivores’ major food is animal tissues, which are lack of toxin, and their bitter taste perception is not so important for their living. Dog, as a first domesticated animal by human, there are less toxic bitter substances in their food and their dependence on bitter perception is decrease compared to its ancestor wolf. Thus, we hypothesize bitter taste receptor genes of dog are under relaxed selection constraints. We studied the microevolution of dog T2R family in the population level and the function of dog T2Rs.We chose 30 dogs from 15 dog breeds and got the 15 members of dog T2Rs, 5 dog T2R pseudogenes and 16 dog intergenic regions of each individual. We first compared the microevolution of T2Rs from dogs and other three placental mammals and then explored the function (binding sites) of dog T2Rs. After population genetic analysis, we first revealed that dog T2Rs has lower genetic diversity than that of intergenic regions. To further investigate the microevolution of different dog T2R genes, we groupes dog T2Rs into three kinds and found multiple to multiple ortholog genes have the highest polymorphism and one to one ortholog gene harbors the minimum polymorphism which indicates different T2R genes are under different selection constraints. This conclusion is consistent with that of other three placental mammals’ T2Rs. The genetic diversity of dog T2R14 is even higher than that of intergenic regions and there are 17 haplotypes among 30 dogs, which shows that it may be under relaxed purifying selection or diversifying selection.To characterize the function of dog T2Rs, we heteroexpressed dog T2Rs by in vitro cell heteroexpressed function assay system and challenged each dog Tas2R with 33 bitter compounds. We reported 5 ligands for 3 dog T2Rs for the first time, which are dTas2R1, dTas2R10 and dTas2R14. The 3 ligands for dTas2R1 are Colchicine, (-)-α-Thujone and 3-Methyl-1-pentyn-3-ol. Diethylene triamine pentaacetic acid (DTPA) is the ligand of dTas2R10 and the ligand of dTas2R14 is 6-Propyl-2-thiouracil (PROP). We challenged PROP structure related compounds with dTas2R14 and found the important chemical structure influenced the activation of dTas2R14. Because there are 17 alleles of dT2R14 and the ortholog gene of human is hT2R43-44 cluster which ligands are known, we conducted the function study of all the 17 dT2R14 alleles to reveal the functional differentiation of dog T2Rs in populations. By heteroexpressed 17 dTas2R14 haplotypes and function assays, we found amarogentin, the second natural ligand of dTas2R14. The response of amarogention is the same with that of PROP. Furthermore, we found the 17 dTas2R14 variations are divided into ‘functional’ and ‘nonfunctional’ types, 13 variations can be activated by the two ligands while the left 4 haplotypes can not. The amino acid site 91 of all the functional variations is serine (S) while that of all the nonfunctional haplotypes is asparagine (N). By the functional experiment of mutated dTas2R14 variations, we found amino acid site 91 is the critical site for the activation of dTas2R14. Additionally, we found another two amino acid sites, site 175 and site 185, also influence the function of dTas2R14. This research will provide further understanding of the relationship of T2R genes variations and the receptor function divergence. |
| 语种 | 中文 |
| 公开日期 | 2013-06-06 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7453]  |
| 专题 | 昆明动物研究所_进化与功能基因组学 |
| 推荐引用方式 GB/T 7714 | 胡玲玲. 家犬苦味受体基因族的进化和功能研究[D]. 北京. 中国科学院研究生院. 2013. |
入库方式： OAI收割
来源：昆明动物研究所
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