部分禽类mtDNA序列测定及在不同来源禽肉鉴定上的应用

发布时间：2018-05-29 06:07

本文选题：鸿雁 + 斑嘴鸭　；参考：《扬州大学》2015年硕士论文

【摘要】：目前市场上肉类品种混杂,掺杂掺假、以次充好的现象屡禁不止。针对这些现象,亟需建立一种快速的检测技术。线粒体基因组(mtDNA)具有多态性丰富、无重组、母系遗传等特点,是开展系统发育学、分子生态学、分类学等研究的理想分子标记。我国家禽品种众多,某一家禽品种难以代表本物种的遗传特征,本研究以线粒体基因作为目标基因,在获取鸿雁(Anas poecilorhyncha,中国鹅种的祖先)和斑嘴鸭(Anas poecilorhyncha,家鸭祖先之一)线粒体全序列基础上,选取细胞色素氧化酶基因Ⅰ(COI)作为目标基因,对6种具显著代表性的家禽肉(鸡、鸭、鹅、鹌鹑、火鸡和鸽)线粒体DNA基因条形码区域进行扩增与鉴定,通过序列比对等方法建立了一种快速鉴别禽肉的分子生物学方法。主要研究结果如下：通过对斑嘴鸭线粒体基因组全序列的扩增,获得了斑嘴鸭线粒体全序列16 606bp,并提交Genbank (AccessionNo.KF751616),其包含22个tRNA、2个rRNA基因、13种蛋白质编码基因和1个D-loop区。碱基组成T为22.2%,C为32.8%,A为29.2%,G为15.8%,无明显的AT偏好性,22种tRNA都为典型的三叶草结构,参照棕头鸦(Larus brunnicephalus)和黑尾地鸦(Podoces hendersoni)的12S rRNA,对斑嘴鸭12S rRNA的二级结构进行了预测,含有4个结构域和37个茎环。对D-loop控制区序列分析发现其含有goose hairpin, E-box, F-box, D-box, Bird similarity-box及CSBl-box。以原鸡作为参考群体,采用NJ算法和ML算法,基于线粒体基因组全序列及D-loop区(鸭属4个种,家鸭3个种,原鸡)分别构建系统进化树,发现3个家鸭品种与绿头鸭亲源关系较近。通过同样的方法,我们获得了鸿雁16 739 bp的线粒体全序列,并提交GenBank (AccessionNo.KJ124555),其包含22个tRNA、2个rRNA基因、13种蛋白质编码基因和一个D-loop区。碱基组成T为22.49%,C为32.24%,A为30.21%,G为15.06%,无明显的AT偏好性。22种tRNA都为典型的三叶草结构,参照原鸡(Gallus gallus)和黑尾地鸦(Podoces hendersoni)的12S rRNA,对鸿雁12S rRNA的二级结构进行了预测,发现其含有4个结构域、37个茎环和13个突出部。对D-loop控制区序列分析发现,含有LSP/HSP、 ETAS1-2、Goose hairpin、E-box、F-box、D-box、C-box、Bird similarity-box、CSB1-box、 CSB-like和OH。同样以原鸡作为参考群体,采用邻接法(NJ)、最大拟然法(ML)算法和贝叶斯法,基于线粒体基因组全序列分别构建系统进化树,发现鸿雁与灰雁(Anser anser)、豆雁(Anser fabalis)、白额(Anser albifrons)和加拿大黑雁(Branta canadensis)处于一个分支,亲缘关系较近。通过对鸡雁小纲(Galloanserae)线粒体基因组全序列进行分析,筛选出COI基因上游基因条形码区域作为候选目标区域,并针对其两端保守区域设计禽类通用引物(P4),并以发表的鸟类COI通用引物(P8)为对照,以鸡、鸭、鹅、鹌鹑、火鸡和鸽六种禽肉为实验材料,采用PCR-RFLP对6种不同来源禽肉的COI条形码区域进行了扩增与鉴定,试验结果显示,6种禽类在P4通用引物扩增的酶切产物中均呈现出不同的条带组合,P8通用引物可扩增出不同的条带组合,由此可见,P4和P8两种COI条形码通用引物都可实现对6不同禽肉准确的鉴定。
[Abstract]:At present, the market of meats is mixed, adulterated and adulterated, and the phenomenon is repeatedly forbidden. In view of these phenomena, it is urgent to establish a rapid detection technique. The mitochondrial genome (mtDNA) has the characteristics of rich polymorphism, no recombination and maternal inheritance. It is an ideal molecular marker for the development of systematic development, molecular ecology, taxonomy and so on. There are many species of poultry in our country, and a poultry breed is difficult to represent the genetic characteristics of this species. This study uses mitochondrial gene as the target gene to select the cytochrome oxidase gene on the basis of the complete mitochondrial sequence of the Anas poecilorhyncha, the ancestor of the Chinese goose and the Anas poecilorhyncha, one of the ancestors of the domestic duck. I (COI) was used as the target gene to amplify and identify the 6 typical poultry meat (chicken, duck, goose, quail, Turkey, and pigeon) in the barcode region of the mitochondrial DNA gene. A molecular biologic method for rapid identification of poultry was established by sequence comparison. The main results were as follows: through the mitochondrial gene of duck mouth duck Group full sequence amplification, obtained the full sequence of 16 606bp, and submitted Genbank (AccessionNo.KF751616). It contains 22 tRNA, 2 rRNA genes, 13 protein coding genes and 1 D-loop regions. The base composition T is 22.2%, C is 32.8%, A is 29.2%, G is 15.8%, no obvious AT preference, 22 tRNA are typical clover structure. With the reference to the 12S rRNA of Larus brunnicephalus and Podoces hendersoni, the two stage structure of 12S rRNA in speckled duck was predicted, containing 4 domains and 37 stem rings. The reference group, using the NJ algorithm and the ML algorithm, constructed the phylogenetic tree based on the complete mitochondrial genome sequence and the D-loop region (4 species of ducks, 3 species of domestic ducks, and the chicken), and found that 3 domestic duck breeds were closely related to the parent source of green duck. By the same method, we obtained the complete mitochondrial sequence of Hongyan 16739 BP and submitted GenBank (Access). IonNo.KJ124555), which consists of 22 tRNA, 2 rRNA genes, 13 protein coding genes and one D-loop region. The base composition T is 22.49%, C is 32.24%, A is 30.21%, G is 15.06%, and no obvious AT preference.22 tRNA are typical three leaf clover structures. The two level structure of 2S rRNA was predicted, and it was found to contain 4 domains, 37 stem rings and 13 protrusions. The sequence analysis of D-loop control region found that it contains LSP/HSP, ETAS1-2, Goose hairpin, E-box, F-box, D-box, C-box, Bird, as the reference group, and the largest The ML algorithm and Bias Fa, based on the complete sequence of mitochondrial genome, constructed the phylogenetic tree respectively, and found that Hongyan and gray goose (Anser anser), Anser fabalis (Anser fabalis), white amount (Anser albifrons) and Canadian wild goose (Branta canadensis) are in a branch and close relationship. Through the mitochondrial base of chicken goose small class (Galloanserae) According to the whole sequence analysis, the COI gene barcode area was selected as the candidate target area, and the common primer (P4) was designed for the conservative regions at both ends. And the published COI universal primers (P8) of the birds were used as the control. The six kinds of poultry meat, chicken, duck, goose, quail, Turkey and pigeon, were used as experimental materials, and 6 different kinds were used by PCR-RFLP. The COI barcode area of the source poultry was amplified and identified. The results showed that 6 kinds of birds showed different bands in the P4 universal primers, and the P8 universal primers could amplify the different band combinations. Thus, the P4 and P8 two COI bar code general primers can realize the accurate identification of 6 different poultry meat. Make sure.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.2

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