摘要
线粒体DNA(mtDNA)的异质性(heteroplasmy)是多种不同序列的mtDNA拷贝共存于一个个体的现象,对线粒体、细胞和组织的机能产生明显的影响。粪便是野生动物研究中最易得的材料,含有动物肠上皮脱落的细胞及其DNA,其中的mtDNA异质性可以反映个体在各种因素影响下肠上皮细胞DNA的稳定性,为揭示个体的适合度、生态对策等提供新的线索。梅花鹿(Cervus nippon)是森林生态系统中的关键物种,其肠上皮中mtDNA异质性的特点尚未见报道。本研究用27只6~96月龄的圈养梅花鹿粪便样本,采用PCR扩增子高通量测序技术,对mtDNA D-loop区的异质性进行定量分析。结果显示:D-loop区广泛分布着异质性位点,但相对集中于第175~368位碱基的高变区(HyperHPR),而第597~687位碱基区间则较少发生,为保守区(Hy⁃poHPR)。HyperHPR异质性突变的多样性(HDα)极显著高于HypoHPR(P<0.001)。转换(T_(S))是主要碱基替换类型,其发生频率远高于颠换(T_(V))。HyperHPR中T_(S)/T_(V)平均为0.11,而HypoHPR平均为0.19,说明异质性突变在HypoHPR受到更强的选择性清除。无论是在D-loop区、HyperHPR与HypoHPR,雌雄个体之间异质性突变指标均无显著差异(D-loop区,|t|=0.273,P=0.787;HyperHPR,|t|=0.409,P=0.686;Hy⁃poHPR,|t|=0.793,P=0.435)。此外,HyperHPR的异质性频率与月龄之间存在显著正相关关系(R^(2)=0.132,P=0.035),使整个D-loop区也具有近乎显著的趋势(R^(2)=0.104,P=0.056)。但是,异质性突变各项指标均与性别无关(P=0.435~0.787)。以上结果表明,肠上皮细胞的D-loop区作为接近中性进化的非编码区,能够保留大量的异质性突变,但对T_(V)突变产生明显的选择性清除,特别是保守区域对T_(S)和T_(V)均有强烈的清除作用,从而使异质性突变形成的mtDNA多样性控制在一个相对正常的范围内。年龄是影响异质性水平的重要因素,性别的影响较小。研究结果为利用粪便研究肠上皮的稳定性与环境的关系提供了新的视角。
The heteroplasmy of mitochondrial DNA(mtDNA)is a phenomenon that multiple variants coexist in an indi⁃vidual.It affects the functions of mitochondria,cells,and tissues.Feces is the most conveniently collectable material from wildlife,containing exfoliated gut epithelial cell and their DNAs.It could be expected that the heteroplasmy of fecal mtDNA could be an indicator of genetic stability of epithelium under various environmental factors,providing new clues into indi⁃vidual fitness and effectiveness of ecological strategies.The sika deer(Cervus nippon)is a key species in forest ecosys⁃tems.The gut epithelial heteroplasmy has not yet been studied.The present study characterized the heteroplasmy in Dloop region of fecal mtDNA using fecal samples of 27 captive sika deer between 6 and 96 months old,and high-through put sequencing of PCR products.The results showed heteroplasmic mutation wide spread throughout the D-loop,but concen⁃trated in the hyperheteroplasmic region(HyperHPR)ranging from the 175th to 368th nucleotide.The region between the 597th and 687th nucleotide was a hypoheteroplasmic region(HypoHPR)where carried very few mutations.Theαdiversity of heteroplasmy diversity(HDα)of HyperHPR was extremely significantly higher than that of HypoHPR(P<0.001).Tran⁃sition(T_(S))was the preponderant type of nucleotide substitution,of which the occurrence was significantly higher than trans⁃version(T_(V)).The ratio T_(S)/T_(V)was averaged 0.11 in HypoHPR and 0.19 in HyperHPR,suggesting the mutations were more selectively removed from HypoHPR.There was no significant difference between males and females for the entire D-loop region(|t|=0.273,P=0.787)and the HyperHPR(|t|=0.409,P=0.686)and HypoHPR(|t|=0.793,P=0.435).Meanwhile,the frequency of heteroplasmy in HyperHPR was significantly correlated with age in month(R^(2)=0.132,P=0.035),leading to a marginal significance in D-loop region(R^(2)=0.104,P=0.056).However,no relatedness was ob⁃served between sex and heteroplasmic indices(P=0.435-0.787).The above results suggest that D-loop region of gut epithelium could accommodate a large quantity of heteroplasmic mutations though it generally evolves neutrally.However,selective elimination acts strongly for T_(V)across the whole region,and even stronger for both T_(S) and T_(V)in HypoHPR,which results in the diversity of heteroplamy laying below an acceptable threshold.Age is an influencing factor of heteroplasmic mutation,while sex is not.In general,this study provides a novel angle to regard the relationship between gut epithelium stability and environmental factors.
作者
高雨薇
李晓达
周永恒
林力均
杨金城
储佳宁
王文慧
张琦
于梦佳
徐艳春
杨淑慧
GAO Yuwei;LI Xiaoda;ZHOU Yongheng;LIN Lijun;YANG Jincheng;CHU Jianing;WANG Wenhui;ZHANG Qi;YU Mengjia;XU Yanchun;YANG Shuhui(College of Wildlife and Protected Area,Northeast Forestry University,Harbin 150040,China;National Forestry and Grassland Administration Research Center of Engineering Technology for Wildlife Conservation and Utilization,Harbin 150040,China)
出处
《野生动物学报》
北大核心
2025年第3期564-572,共9页
CHINESE JOURNAL OF WILDLIFE
基金
国家自然科学基金项目(32170517)。
关键词
梅花鹿
粪便DNA
线粒体异质性
年龄
Sika deer(Cervus nippon)
Fecal DNA
MtDNA heteroplasmy
Age
