鸡线粒体全基因组DNA异质性及其潜在效应

In a long time, mitochondrial heteroplasmy was thought to be rare, and only to be found in patients with a phenomenon referred to as the threshold effect (a minimum number of mutant genomes were required for the expression of disease).The recent study shows that human mitochondrial heteroplasmy is one common phenomenon while not one rare case. Up till now there were no reports about chicken mitochondrial heteroplasmy. In our lab, mitochondrial G4580A variant was found to be a heteroplasmic variation site and have potential important effect on some traits recently. Based on the preliminary findings, we plan to further conduct the following study: ① identify chicken mitochondrial DNA(mtDNA) heteroplasmy from the leg tissue in whole genomic mtDNA level by Solexa high throughput sequencing method; ②Based on the identified mitochondrial heteroplasmic sites, conduct the study about the tissue/breed distribution characteristics of these heteroplasmic sites with allele specific fluorescence quantitative PCR method (ASP-qPCR); ③conduct the study about the transmission characteristics of the allele for these heteroplasmic sites with the Gushi chicken resource population; ④conduct the association analysis between the mtDNA heteroplasmy of these sites and the related traits/ gene expression with F2 individuals of Gushi chicken resource population.Our object was to learn about the chicken mitochondrial DNA heteroplasmy in genomic mtDNA level, the heteroplasmic characteristics and the potential effect, which would make a foundation for the application of mtDNA heteroplasmy on chicken molecular breeding and phylogenetic study.

线粒体异质性长期被认为呈阈值效应，十分稀有，仅出现在相应的病人中。但近年发现，人类线粒体DNA异质性具有普遍性。目前关于鸡线粒体异质性的研究尚未见报道。本课题组已发现鸡线粒体G4580A位点存在异质性并显示了潜在的重要效应。本项目拟在此基础上：①采用Solexa高通量测序，进行鸡腿肌组织线粒体DNA(mtDNA)全基因组异质性的鉴定及特性分析；②对鉴定的异质性变异位点，采用等位基因特异荧光定量PCR(ASP-qPCR)，研究mtDNA异质性的组织/品种分布特性；③利用固始鸡资源群体,研究异质性位点等位基因的传递特性；④利用固始鸡资源群F2 代个体,进行mtDNA异质性与相关性状/相应基因表达量的关联分析。以掌握鸡线粒体基因组异质状况、异质特性及其潜在效应。该研究可为利用mtDNA异质性进行鸡的分子选育和种系发育研究奠定基础。

线粒体异质性具有阈值效应，严重影响人类健康。目前关于鸡线粒体异质性的研究尚少见报道。（1）本研究采用Solexa高通量测序进行鸡线粒体DNA(mtDNA)全基因组点异质性研究，在5‰水平检测到217个点异质性位点，84%为转换，仅16%为颠换；D-Loop 是异质性发生的热点区域，64%的异质性位点位于D-Loop区。除位于D-Loop mt.A683G、mt.C737T和位于Cox2 内mt.G8682A位点具有高异质性外(>30%)，其余位点均为低异质性位点(0.01%~6.65%）；91个点异质性位点也是变异位点，显示异质性可能是变异的一个过渡类型；（2）线粒体插入/缺失异质性的分析表明， 5‰水平共检测到49插入/缺失异质性位点，大部分位点为低异质性程度位点。有20个位点有两种以上等位基因型，D-LOOP区有相对更多的变异类型。（3）全基因组水平下进行不同组织间的点异质性和插入/缺失异质性分析显示，所有异质性位点在不同组织间具有相同的优势等位基因，未发现不同组织间的异质性。绝大部分位点为低异质性位点，仅mt.G8682A呈现了相对高的组织内异质性。（4）采用PCR-RFLP开展了五个具有不同异质性程度的点异质性位点（除mt.G8682A外，mt.A4589G、mt.A5703T、mtT5727G、mt.G16121A在高通量测序中均报道为低异质性位点）变异在品种间的分布特性分析，发现五个位点均检测到了异质性，这进一步显示了高通量测序的灵敏性；此外除mt.G8682A外，其它四个位点在不同品种间具有不同的优势等位基因。（5）与固始鸡资源群性状的关联分析，发现ND1 mt. 4589A>G异质性对腹脂率和皮下脂肪厚度的影响显著 (P<0.05)； ND2 mt.ND2 5703A>T和mt.ND2 5727T>G异质性对胸肌脂肪含量具有显著的效应。（6）利用构建的异质性群体和固始鸡资源群体开展了线粒体变异/异质性的传递特性分析，发现线粒体变异/异质性总体为母系遗传模式，未发现明显的线粒体父系渗漏现象。（7）能量限制对ND2基因的线粒体异质性和基因表达水平均无显著效应，但不同的油脂源对ND2基因的线粒体拷贝数无显著的效应，但显著调控了线粒体ND2和18S rRNA在胸肌和肝脏组织的表达水平。 相关研究为进一步开展鸡线粒体异质性的功能研究奠定了基础。