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莫大颖,江海英,武正军,黄铭威,陈金平.2019.不同地理种群鳄蜥tlr4基因遗传多样性 及SNPs分布特点分析.动物学杂志,54(6):845-859.
不同地理种群鳄蜥tlr4基因遗传多样性 及SNPs分布特点分析
Genetic Diversity and SNPs Distribution Characteristics of tlr4 Gene in Different Geographic Populations of the Chinese Crocodile Lizard
投稿时间:2019-07-04  修订日期:2019-10-24
DOI:10.13859/j.cjz.201906011
中文关键词:  Toll样受体4  遗传多样性  点突变  鳄蜥
英文关键词:Toll-like receptor 4  Genetic diversity  Point mutations  Shinisaurus crocodilurus
基金项目:广州市科技计划项目重点项目专题(No. 201804020080),国家自然科学基金项目(No. 31760623)
作者单位E-mail
莫大颖 ① 珍稀濒危动植物生态与环境保护教育部重点实验室 桂林 541004 ② 广西濒危动物生态学重点实验室 桂林 541004③ 广东省生物资源应用研究所 广州 510260 573741049@qq.com 
江海英 ③ 广东省生物资源应用研究所 广州 510260 874788116@qq.com 
武正军 ① 珍稀濒危动植物生态与环境保护教育部重点实验室 桂林 541004 ② 广西濒危动物生态学重点实验室 桂林 541004 wu_zhengjun@aliyun.com 
黄铭威 ③ 广东省生物资源应用研究所 广州 510260 374115046@qq.com 
陈金平 ③ 广东省生物资源应用研究所 广州 510260 chenjp@giabr.gd.cn 
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中文摘要:
      Toll样受体(TLRs)是一类识别病原体中高度保守的病原相关分子模式(PAMPs)的蛋白家族,在启动先天性免疫中具有重要作用。人类toll样受体4基因(toll4)的点突变与许多疾病有关,如呼吸道合胞病毒感染、动脉粥样硬化、疟疾等,并且这种点突变往往具有种族分布特异性。鳄蜥(Shinisaurus crocodilurus)是一种古老的爬行动物,属于国家Ⅰ级保护野生动物,由于其遭受非法捕杀、栖息地破坏等人为干扰,目前数量极其稀少。近年来,鳄蜥因疾病造成大量的死亡,且这种疾病的分布具有种群差异性,不同种群往往疾病症状也不同,可能是因不同的病原体感染引起的。我们设想toll4基因的点突变在鳄蜥种群之间可能具有分布的差异性,以适应不同的当地病原体。因此,本文利用PCR产物直接测序和生物信息学分析法初步探索鳄蜥toll4基因的遗传多样性以及单核苷酸多态性(SNPs)的分布特点,验证该基因的点突变是否具有种群特异性分布特点,以及预测其氨基酸替换是否对蛋白质结构和功能产生影响。结果获得的扩增片段长为1 694 bp,在5个种群52条序列中(登录号MN380726 ~ MN380777),共发现27个突变位点,导致9个氨基酸替换,其中3个氨基酸替换被预测为影响蛋白质的结构和功能。27个点突变中有12个只分布在单个种群,4个分布无种群特异性,即每个种群中均有野生型和突变型,其余11个点突变的突变型分布在2或3个种群不等。研究表明,鳄蜥toll4基因遗传多态性在各种群间不一致,且点突变具有种群特异性分布特征,反映不同地理种群遭受不同的病原压力。
英文摘要:
      Toll-like receptors (TLRs) are a family of proteins that recognize highly conserved pathogen- related molecular patterns (PAMPs) in pathogens, and play an important role in initiating innate immunity. Point mutations in the human tlr4 gene have been shown to be associated with many diseases, such as respiratory syncytial virus infection, atherosclerosis, malaria, etc., and these mutations are often racially specific. The Chinese Crocodile Lizard (Shinisaurus crocodilurus) is an ancient reptile which belongs to the national first-class protection animal. Due to anthropogenic disturbance, such as illegal hunting and habitat destruction, the number of the Chinese Crocodile Lizard is extremely rare. In recent years, a large number of death of the Chinese Crocodile Lizard has been caused due to various diseases, and the diseases are different among different populations, often with different disease symptoms, which are possibly caused by different pathogens. We predict that point mutations in the tlr4 gene might be distributed differently among the Chinese Crocodile Lizard populations to accommodate different local pathogens. Therefore, the aim of this study is to preliminarily explore the genetic diversity of tlr4 gene and the distribution characteristics of SNPs in the Chinese Crocodile Lizard by using PCR product direct sequencing and bioinformatics analysis, and to verify whether the point mutation on the gene has the population-specific distribution, and to test whether its non-synonymous substitution has an impact on the structure and function of protein. The amplified fragment length was 1 694 bp (Fig. 1), and the genetic diversity of each population was different, with Linzhouding population the highest and Dayaoshan population the lowest, which corresponds to the largest population of Linzhouding and the smallest population of Dayaoshan (Table 1). A total of 27 point mutations were found in 52 individuals (GenBank accession: MN380726﹣MN380777) from five populations, leading to nine non-synonymous substitutions (Table 2), three of which were predicted to affect the function and structure of proteins (Table 4). Twelve of the 27 point mutations were distributed in only one population, while four point mutations were not specific to any population, with wild-types and mutant types in each population, and the other eleven point mutations were distributed in two to three populations (Table 3). Only three of the 31 haplotypes were shared among populations, while the rest were distributed in a single population (Fig. 2). The results have shown that the genetic polymorphism of tlr4 gene is not consistent among different populations, and point mutations have population-specific distribution characteristics, reflecting the fact that different geographical populations are affected by different pathogenic pressures. It is suggested that attention should be paid to the maintenance of genetic diversity in future conservation of this species.
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