Rapid and Sensitive Identification of Horse and Donkey Meat in Iraqi Markets Using SSR and PCR-RFLP Based on Mitochondrial DNA Cytochrome B Gene

This study was carried out at the Genetic Engineering Lab, College of Agriculture, University of Basrah. DNA was extracted from samples containing meat of commercial products in the Basrah markets. The products subjected to analysis using polymerase chain reaction （PCR） with species specific repeat （SSR） and polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） techniques based on the sequence of the mitochondrial DNA cytochrome b gene （mtDNA cyt b gene）. Then digestion with the Alu l restriction enzyme to establish a differential diagnosis detects and discriminates between meat species and adulteration in the products. SSR primers were applied, has been detected amplification of the encoded gene product, generated 221 bp in some imported minced and canned meat samples. The results show that SSR analysis produced a pattern that allowed a direct identification of horse and donkey meats in some imported minced and canned meat samples （Hana, Monde and Bavaria）. The amplified 359 bp gene of mtDNA cyt b gene from samples in different product was cut using Alu 1 restriction enzyme resulting in restriction fragment length polymorphism （RFLP）. Alu 1 was used to distinguish between the animals meat that belong to the family or one species. The digestion of the PCR product showed differences between products. Where the fragment length generated were 74, 76 and 189 bp. It belonged to horse meat. The fraud was detected in Hana, Monde and Bavaria products available in Basrah markets showing the presence of horse meat in these products that labeled as beef meats 100%. This revealed mtDNA cyt b gene as highly conserved and consequently a good molecular marker for diagnostic studies. Thus, this technique can be applied to food authentication for the identification of different species of animals in food products.     

线粒体标记分析安徽省三个家鸭群体间遗传多样性及遗传关系

本研究通过mtDNA序列多样性分析了安徽省地方鸭种巢湖鸭和枞阳媒鸭两个类群的遗传多样性及遗传关系.通过对巢湖鸭、枞阳媒鸭保种群体和枞阳媒鸭怀宁群体3个群体共计85个个体进行mtDNA D-loop区610 bp序列进行测序,共获得了24种单倍型.分析结果表明:巢湖鸭单倍型多样度和核苷酸多样度分别为0.756和0.01115,遗传多样性最高,枞阳媒鸭保种群体次之,分别为0.552和0.00176,枞阳媒鸭怀宁群体遗传多样性最低,分别为0.515和0.00140.系统进化分析结果表明:巢湖鸭与枞阳媒鸭两个群体间遗传关系较远,枞阳媒鸭两个群体间遗传关系最近,但是出现了群体分化的趋势.本研究中枞阳媒鸭两个群体由于其地域差异以及后期进行人工选择导致这两个群体有较大的差异,可以作为两个类群进行研究和开发利用.     

家蚕微孢子虫NbTom40的原核表达及定位

线粒体外膜易位酶是由Tom40基因编码的位于线粒体外膜上的转位酶复合体,能够将外界蛋白质转位运输到线粒体基质中.最近有研究称在一类仅具有简缩进化的线粒体——纺锤剩体的感染家蚕微孢子虫的基因组鉴定到一个NbTom40基因.本文首先通过克隆了长度为839 bp的NbTom40基因,通过构建重组载体pET30a(+)-NbTom40,转入E.coli BL21(DE3)感受态细胞中进行诱导表达,经过HIS抗体的免疫印迹对表达的目的蛋白——31～43 kDa间存在一条单一条带进行验证,结果显示该条带即为分子量大小正确的带有HIS标签的目的蛋白,然后将其进行Ni-NTA亲和层析以及聚丙烯酰胺凝胶纯化并制备多克隆抗体.NbTom40抗体与家蚕微孢子虫总蛋白的Western Blotting结果显示,在32 kD处有明显杂交信号,与理论分子大小一致.最后,通过间接免疫荧光(IFA)对该基因编码的蛋白进行细胞定位,观察到NbTom40以散点状荧光信号分布在孢子内部.本研究为深入了解纺锤剩体的蛋白组成及其膜转运的分子机制奠定了分子生物学基础.     

不同地理群体的鲇线粒体DNA 16S rRNA基因序列比较分析

对长江中上游主要支流5个野生群体鲇[舞阳河群体(WYH)、乌江群体(WJ)、雅砻江群体(YLJ)、岷江群体(MJ)、金沙江群体(CS)]中的27个样品的16S rRNA基因进行PCR扩增,并对扩增产物测序.用CLUSTAL X v 1.81软件对所得的27个16SrRNA序列进行比对,并用Dna SP 5.10软件进行比较分析,共检测出74个变异位点、8种单倍型.舞阳河、乌江、雅砻江、岷江、金沙江等5个野生群体的单倍型多样性(H)分别为0.600、0.857、0.600、0.600、0.667,核苷酸多样性(π)分别为0.00072、0.1754、0.00036、0.00072、0.01871.对5个野生群体的16S rRNA序列进行Tajima's D和Fst分析发现,所有群体符合中性进化模型,且有一些单倍型的分化,只有金沙江群体的遗传差异显著.对5个群体进行分子变异等级分析的结果表明,群体间分子变异不显著.对5个群体构建分子系统树发现,乌江、雅砻江、岷江、金沙江群体之间的亲缘关系较近,舞阳河群体与其他群体间的亲缘关系较远.     

姬松茸多糖诱导HepG2 细胞凋亡的线粒体通路研究

为探讨姬松茸多糖诱导HepG2细胞凋亡过程中线粒体膜电位和细胞内Ca2+浓度([Ca2+]i)的变化.以人肝癌细胞系HepG2细胞为对象,使用流式细胞仪检测姬松茸多糖对HepG2细胞凋亡及线粒体膜电位的影响,激光共聚焦显微镜检测HepG2细胞[Ca2+]i的变化.结果表明姬松茸多糖能显著诱导HePG2细胞凋亡,并不同程度降低HepG2细胞线粒体跨膜电位,升高细胞[Ca2+]i,造成钙稳态失衡.姬松茸多糖诱导HepG2细胞凋亡的机制与降低线粒体膜电位,造成细胞内Ca2+超载有关.     

基于线粒体基因组的穿山甲系统发育研究

利用已测定的中华穿山甲线粒体基因组全序列,结合从GenBank中获得的30种哺乳纲动物的线粒体基因组全序列,以针鼹（Tachyglossus aculeatus）和鸭嘴兽(Ornithorhynchus anatinus)为外群,以最大简约法(MaximumParsimony,MP)和贝叶斯推论法(Bayesian Inference,BI)重建了分子系统树。结果表明,在重建的MP和BI系统树中,中华穿山甲和食肉目为姐妹支,支持中华穿山甲和食肉目为姐妹群的观点。     

线粒体凋亡途径在硫酸黏菌素致PC12细胞凋亡中的作用

探讨线粒体凋亡途径在硫酸黏菌素诱导PC12细胞凋亡中作用.取对数生长期PC12细胞,用含0、62.5、125、250 μg· mL-1硫酸黏菌素DMEM培养液,作用24 h,采用Hoechst33258荧光染色法检测PC12细胞凋亡,Western blot法检测细胞色素C(Cyt-C)、Bax、Bcl-2蛋白表达含量,试剂盒检测Caspase-3活性.与对照组相比,125、250 μg· mL-1硫酸黏菌素剂量组细胞PC12细胞Cyt-C、Bax蛋白表达量、Caspase-3活性显著升高(P＜0.01);Bcl-2蛋白表达显著降低(P＜0.01),具有剂量-效应关系.说明线粒体途径介导PC12细胞凋亡是硫酸黏菌素神经毒性作用机制之一.     

A Genetic Diversity Analysis of Mitochondrial DNA D-loop Region in Four High Quality Chicken Breeds

This study was conducted to elucidate the genetic diversity of mitochondrial DNA (mtDNA) D-loop region in Qingyuan partridge chicken group 1,Qingyuan partridge chicken group 2,Yangshan chicken and Qingyuan Yellow feather black-bone chicken.The specific primers were designed according to mtDNA D-loop region of Gullus gullus spadiceus (accession No.:NC_007235.1) in GenBank.The sequence was analyzed after PCR amplification and sequencing,and the haplotype number,polymorphism number,haplotype diversity,nucleotide diversity and nucleotide mean difference were counted.The evolution divergence among breeds was calculated by Mega 5.10 software,and the phylogenetic tree was constructed.The results showed that the length of mtDNA D-loop region in four high quality chicken breeds was 591 bp,and 549 bp were used for subsequent analysis.The content of A,T,C and G were 27.2% to 27.3%,30.1% to 30.4%,29.5% to 29.8% and 12.8% to 12.9%,respectively,and the average content of G+C was 42.5%.There were 92 polymorphic sites which contained 14 singleton variable sites and 78 parsimony informative sites,and the percentage of transitions and transversions were 89.13% (82/92) and 10.87% (10/92),respectively.The haplotype diversity ranged from 0.682 to 0.835,and the nucleotide diversity ranged from 0.00849 to 0.01167.There were 32 haplotypes in all sequences,which could be divided into clades A,B,C and E,however,most of the individuals belonged to clades B (51.2%) and E (37.6%).The phylogenetic tree results showed that four high quality chicken breeds could be classified as 4 branches which were consistent with the haplotypes classification results.The results indicated that the four high quality chicken populations from Qingyuan had relatively high haplotype and nucleotide diversity and likely shared two or more common maternal lineages.