本文選題：鴻雁 + 斑嘴鴨　； 參考：《揚州大學(xué)》2015年碩士論文

【摘要】：目前市場上肉類品種混雜,摻雜摻假、以次充好的現(xiàn)象屢禁不止。針對這些現(xiàn)象,亟需建立一種快速的檢測技術(shù)。線粒體基因組(mtDNA)具有多態(tài)性豐富、無重組、母系遺傳等特點,是開展系統(tǒng)發(fā)育學(xué)、分子生態(tài)學(xué)、分類學(xué)等研究的理想分子標(biāo)記。我國家禽品種眾多,某一家禽品種難以代表本物種的遺傳特征,本研究以線粒體基因作為目標(biāo)基因,在獲取鴻雁(Anas poecilorhyncha,中國鵝種的祖先)和斑嘴鴨(Anas poecilorhyncha,家鴨祖先之一)線粒體全序列基礎(chǔ)上,選取細胞色素氧化酶基因Ⅰ(COI)作為目標(biāo)基因,對6種具顯著代表性的家禽肉(雞、鴨、鵝、鵪鶉、火雞和鴿)線粒體DNA基因條形碼區(qū)域進行擴增與鑒定,通過序列比對等方法建立了一種快速鑒別禽肉的分子生物學(xué)方法。主要研究結(jié)果如下：通過對斑嘴鴨線粒體基因組全序列的擴增,獲得了斑嘴鴨線粒體全序列16 606bp,并提交Genbank (AccessionNo.KF751616),其包含22個tRNA、2個rRNA基因、13種蛋白質(zhì)編碼基因和1個D-loop區(qū)。堿基組成T為22.2%,C為32.8%,A為29.2%,G為15.8%,無明顯的AT偏好性,22種tRNA都為典型的三葉草結(jié)構(gòu),參照棕頭鴉(Larus brunnicephalus)和黑尾地鴉(Podoces hendersoni)的12S rRNA,對斑嘴鴨12S rRNA的二級結(jié)構(gòu)進行了預(yù)測,含有4個結(jié)構(gòu)域和37個莖環(huán)。對D-loop控制區(qū)序列分析發(fā)現(xiàn)其含有g(shù)oose hairpin, E-box, F-box, D-box, Bird similarity-box及CSBl-box。以原雞作為參考群體,采用NJ算法和ML算法,基于線粒體基因組全序列及D-loop區(qū)(鴨屬4個種,家鴨3個種,原雞)分別構(gòu)建系統(tǒng)進化樹,發(fā)現(xiàn)3個家鴨品種與綠頭鴨親源關(guān)系較近。通過同樣的方法,我們獲得了鴻雁16 739 bp的線粒體全序列,并提交GenBank (AccessionNo.KJ124555),其包含22個tRNA、2個rRNA基因、13種蛋白質(zhì)編碼基因和一個D-loop區(qū)。堿基組成T為22.49%,C為32.24%,A為30.21%,G為15.06%,無明顯的AT偏好性。22種tRNA都為典型的三葉草結(jié)構(gòu),參照原雞(Gallus gallus)和黑尾地鴉(Podoces hendersoni)的12S rRNA,對鴻雁12S rRNA的二級結(jié)構(gòu)進行了預(yù)測,發(fā)現(xiàn)其含有4個結(jié)構(gòu)域、37個莖環(huán)和13個突出部。對D-loop控制區(qū)序列分析發(fā)現(xiàn),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。同樣以原雞作為參考群體,采用鄰接法(NJ)、最大擬然法(ML)算法和貝葉斯法,基于線粒體基因組全序列分別構(gòu)建系統(tǒng)進化樹,發(fā)現(xiàn)鴻雁與灰雁(Anser anser)、豆雁(Anser fabalis)、白額(Anser albifrons)和加拿大黑雁(Branta canadensis)處于一個分支,親緣關(guān)系較近。通過對雞雁小綱(Galloanserae)線粒體基因組全序列進行分析,篩選出COI基因上游基因條形碼區(qū)域作為候選目標(biāo)區(qū)域,并針對其兩端保守區(qū)域設(shè)計禽類通用引物(P4),并以發(fā)表的鳥類COI通用引物(P8)為對照,以雞、鴨、鵝、鵪鶉、火雞和鴿六種禽肉為實驗材料,采用PCR-RFLP對6種不同來源禽肉的COI條形碼區(qū)域進行了擴增與鑒定,試驗結(jié)果顯示,6種禽類在P4通用引物擴增的酶切產(chǎn)物中均呈現(xiàn)出不同的條帶組合,P8通用引物可擴增出不同的條帶組合,由此可見,P4和P8兩種COI條形碼通用引物都可實現(xiàn)對6不同禽肉準(zhǔn)確的鑒定。
[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.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.2

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