鼠兔leptin蛋白分子进化特征及其生态学意义

NWIPB OpenIR

	鼠兔leptin蛋白分子进化特征及其生态学意义
其他题名	Characteristic of molecular evolution of leptin protein of pikas (Ochotona) and its ecological significance
	杨洁
学位类型	博士
导师	赵新全
	2007-06-09
学位授予单位	中国科学院西北高原生物研究所
学位授予地点	西北高原生物研究所
关键词	青藏高原适应高原鼠兔 Leptin 适应性功能进化 Rt-pcr Race Real Time Pcr 正向选择
摘要	环境压力可以加速特定压力敏感蛋白的进化速率，创建针对这种特定环境下的新的功能或原有功能的加强，从而提高动物对这种压力环境的适合度。青藏高原独特的气候环境对生活于其中的各种生物的分布、种群结构、生存适应、进化模式等产生深刻的影响。鼠兔作为现代全北区特有物种，呈现出特殊的分布现象：在全球现存的26种鼠兔中，除3种之外，其余均分布于亚洲，其中大部分分布于青藏高原，占全球鼠兔种类的80%以上，且几乎所有鼠兔都分布于高海拔或高纬度的寒冷地区。其中高原鼠兔是青藏高原的关键种和特有种，对维护青藏高原生态系统平衡起到重要作用。高氧利用率、高基础代谢率、高产热能力是高原鼠兔对高寒低氧高原环境最明显的适应特征。Leptin是一种主要由白色脂肪组织分泌的激素，在维持机体能量平衡中起到关键作用。为了揭示鼠兔对高寒低氧高原环境适应的分子机理，本研究采用RT-PCR和RACE方法克隆出高原鼠兔ob基因的全长cDNA序列，并分析了序列特征，同时采用real time PCR方法检测了高原鼠兔ob基因mRNA在不同组织以及不同海拔下的表达特征，并且采用分子进化分析方法分析了鼠兔家族ob基因的分子进化特征。主要研究结果如下： 1. 高原鼠兔ob基因全长cDNA序列由3015bp组成，其编码区序列由504bp组成，位于66-569bp之间。其5’非翻译区（5’UTR）由65bp组成，3’非翻译区（3’UTR）由2446bp组成。起始密码子为ATG，终止密码子为TGA。3’UTR含有PolyA尾，其上游13bp处含有一个加尾信号AAAAT。 2. 高原鼠兔ob基因编码一个由167个氨基酸组成的蛋白质，其N端含有一个21个氨基酸组成的信号肽，因此成熟分泌蛋白由146个氨基酸组成，其预测的分子量为16.086kDa，等电点为6.3。 3. 预测的高原鼠兔leptin蛋白序列中含有一个N-糖基化位点，五个酪蛋白激酶Ⅱ磷酸化作用位点（Casein Kinase Ⅱphosphorylation site），一个蛋白激酶C磷酸化作用位点（protein kinase C phophorylation site）和一个ATP合成酶α和β亚基识别位点（synthase α and β subunits signature），预测其二级及三级空间结构与其它物种leptin分子结构相似，为四股双螺旋组成的束状分子。 4. 高原鼠兔ob基因编码区核苷酸序列与猪、狗、牛、猫、人、鼠同源性分别为83%、82%、81%、81%、81%和79%，推导的氨基酸序列同源性达70～72%，且与其它物种氨基酸序列比对发现高原鼠兔所特有的20个变异位点。 5. 高原鼠兔ob基因可以在脑、心、肺、肝脏、脾脏、肾脏、骨骼肌、脂肪组织中表达，其中以白色脂肪组织中的表达量最多，其次是心脏和肺，而肝脏和肾脏中ob基因的表达量最低，这种多组织的表达特征可能对维持高原鼠兔体内leptin的高水平起到调节作用。 6. 随着海拔的增加，高原鼠兔体内ob基因的表达量增加，提示ob基因可能是不同海拔下高原鼠兔体内能量代谢调节的一个重要分子。 7. 鼠兔家族ob基因进化分析结果显示，leptin蛋白对冷环境压力较为敏感，可以看作为寒冷压力反应蛋白。 8. 寒冷的环境压力是驱动冷适应动物鼠兔leptin发生适应进化的重要因子，这种leptin的适应性功能进化有利于提高鼠兔在压力环境下的生存的适合度。 9. 理论上确定了鼠兔leptin蛋白中20个正向选择位点（即适应性功能进化位点）分别为：2S, 4W, 7R, 28H, 29A, 36I, 44A, 59V, 60L, 62K, 63H, 92A, 94Q, 95G, 98P, 103D, 106S, 108N, 111E, 113I。 10. 这种leptin的适应性进化特征可能是鼠兔家族所共有的现象。基于高原鼠兔leptin克隆，cDNA序列以及蛋白质序列结构分析及其mRNA的组织特异性表达、不同海拔下mRNA表达的研究分析，以及鼠兔家族leptin分子进化分析结果，推测鼠兔leptin蛋白可能发生了适应性的功能进化，其功能有别于人、大鼠、小鼠、家兔等其他动物的leptin的功能，在维持鼠兔对极端高原环境压力下的生态适应中起到重要作用。
其他摘要	Environmental stress can accelerate the evolutionary rate of specific stress-response proteins and creates new functions specialized for different individual living environments, enhancing organism's fitness to stressful environments. The Qinghai-Tibet plateau, a unique geographic unit that is the highest plateau in the world has a great impact on the distribution, structure, function, adaptation, and evolutionary patterns of various organisms. Pikas (order Lagomorpha), an endemic, non-hibernating mammal in the modern Holarctic Region, live in cold regions at either high altitudes or high latitudes and have a distribution of maximum species diversification confined only to the Qinghai-Tibet Plateau. Most of the species are confined to those regions with either high altitudes or high latitudes and live in cold climates. Plateau pika (Ochotona curoniae), an endemic species of Qinghai-Tibet Plateau, is known as a keystone species on Qinghai-Tibet Plateau ecosystem and plays an important role in preservation of native biodiversity. High ratio of oxygen utilization, high resting metabolic and high non-shivering thermogenesis is obvious adaptive characteristic for pikas living under harsh plateau environment. Leptin, an adipocyte-derived hormone, plays an important role in body energy homeostasis. To reveal the molecular mechanism of ecological adaptation of plateau pika, in this study, the full-length cDNA of plateau pika leptin was cloned using RT-PCR and RACE. The tissue expression and different altitude distribution of plateau pika leptin mRNA was detected by real time PCR. We also analyzed the characteristic of molecular evolution of ob gene within pika family. The main achievement of this study is listed as follows: 1. The complete leptin cDNA included a coding sequence of 504bp, 5’untranslated region (UTR) of 65bp and 3’UTR of 2446bp. The open reading frame (ORF) initiated with an ATG codon at nucleotide position 66 and terminated with a TGA stop codon at nucleotide position 569. The 3’UTR contained a polyadenylation signal (AAAAT) located 13 bases upstream from the poly(A) tail. 2. The deduced amino acid sequence of plateau pika leptin cDNA was composed of 167 amino acids and encoded an apparent signal peptide sequence of 21 amino acids. Thus, the mature excreted protein has a predicted molecular weight of 16.086 kDa and a pI of 6.3. 3. Motifs which were predicted in deduced amino acid sequence of pika leptin contained one N-glycosylation site, five Casein kinase II phosphorylation sites, one protein kinase C phosphorylation site and one ATP synthase α and β subunits signature site. The pika leptin protein was estimated to comprise four helixes in secondary and third structure similar to that of leptins in other species. 4. The coding sequence of pika leptin cDNA shared 83%, 82%, 81%, 81%, 81% and 79% nucleotide sequence homology to that of pig, dog, cow, cat, human and mouse, respectively. The deduced amino acid sequence of the mature pika lepitn was 70-72% identical to that of other species mentioned above. 5. ob mRNA was expressed in brain, heart, lung, liver, spleen, kidney, muscle and adipose tissues. The tissular specificity was displayed with the highest in white adipose, next in heart and lung, and much less in kidney and liver. This expression characteristic of multi-tissues may be play an important role in maintaining high level of leptin in plateau pika. 6. Comparison of ob gene expression in the two altitudes showed that the expression level of pika ob gene in higher altitude is significantly higher then that in lower altitude, which indicated that leptin may be an important molecular of energy regulation of plateu pika under different altitudes. 7. The result of evolution of ob gene within pika family showed that leptin was sensitive to cold environment and is a cold-stress response protein. 8. Cold stress drives the adaptive functional evolution of leptin within the native cold-adaptive Ochotona family, contributing to fitness enhancement for pikas’ survival in a stressful environment. 9. We identified 20 positive selection(adaptive function evolution)sites as follows: 2S, 4W, 7R, 28H, 29A, 36I, 44A, 59V, 60L, 62K, 63H, 92A, 94Q, 95G, 98P, 103D, 106S, 108N, 111E, 113I. 10. Adaptive functional evolution of pika leptin may be a common characteristic of the entire Ochotona family throughout the world. Granding on the plateau pika leptin cloning, characteristic and mRNA expression in different tissue and different altitudes, and molecular evolution of leptin within pika family, we deduced that positive selection acts on pika leptin, namely, adaptive evolution occurs in pika leptin. The function of pika leptin may be different from that of other species such as human, rat, mouse or rabbit, etc. We concluded that the new adaptive functional evolution of pika leptin may play an important role in this animal’s ecological adaptation to extremely environmental stress.
页数	119
语种	中文
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/3040
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	杨洁. 鼠兔leptin蛋白分子进化特征及其生态学意义[D]. 西北高原生物研究所. 中国科学院西北高原生物研究所,2007.

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